Modified chaperonin 10

ABSTRACT

A novel granule dispersion composition which comprises water and, dispersed therein, a finely pulverized, sparingly water-soluble substance. The granule dispersion composition is prepared by dispersing in water a granular material which comprises a specific substance sparingly soluble in water, a polymer, and an oil and has an average particle diameter of 1 μm or smaller and in which the ratio of the total weight of the polymer and oil to the weight of the specific substance is 1.5 or higher.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a U.S. National Stage Application ofPCT/AU2006/001278 filed Aug. 31, 2006, which claims priority toAustralian Application No. 2005904765 filed Aug. 31, 2005, the contentsof which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to chaperonin 10 polypeptides, and tonucleic acids encoding the same. The present invention further relatesto chaperonin 10 polypeptides displaying immunomodulatory activity, tomethods using the same and to compositions comprising such polypeptides.

BACKGROUND ART

Mammalian chaperonin 10 (Cpn10), also known as heat shock protein 10(Hsp10) and early pregnancy factor (EPF), is typically characterized asa mitochondrial ‘molecular chaperone’ protein involved in proteinfolding together with chaperonin 60 (Cpn60; Hsp60). Cpn10 and Cpn60 arehomologues of the bacterial proteins GroES and GroEL respectively. GroESand Cpn10 each oligomerise into seven member rings that bind as a lidonto a barrel-like structure comprising fourteen GroEL or seven Cpn60molecules respectively, which tether denatured proteins to the complex(Bukau and Horwich, 1998, Cell 92:351-366; Hartl and Hayer-Hartl, 2002,Science 295:1852-1858).

Cpn10 proteins are highly conserved across species, Human Cpn10 is 100%identical to bovine Cpn10 and differs from rat Cpn10 at only a singleamino acid position. Human Cpn10 shares 30% sequence identity (60%similarity) with GroES from Escherichia coli. As illustrated by theheptameric crystal structure of E. coli GroES (see FIG. 1A; Xu et al.,1997, Nature 388:741-750). Cpn10/GroES proteins are comprised ofessentially three different structural regions, an anti-parallelβ-barrel region which is flanked by a “roof” β-hairpin loop region and a“mobile loop” region. The mobile loop region mediates interaction withCpn60/GroEL and is thus critical for the formation of the complex withCpn60/GroEL and for the ‘molecular chaperone’, protein folding activity.

However in addition to its intracellular role as a molecular chaperone,Cpn10 is also frequently found at the cell surface (see Belles et al.,1999, Infect Immun 67:4191-4200) and in the extracellular fluid (seeShin et al., 2003, J Biol Chem 278:7607-7616) and is increasingly beingrecognized as a regulator of the immune response. For example, Cpn10 hasbeen demonstrated to have immunosuppressive activity in experimentalautoimmune encephalomyelitis, delayed type hypersensitivity andallograft rejection models (Zhang et al, 2003, J Neurol Sci 212:37-46;Morton et al, 2000, Immunol Cell Biol 78:603-607).

The present inventors have also recently demonstrated that Cpn10 caninhibit LPS-induced activation of NF-κB, reduce LPS-induced TNFα andRANTES secretion and enhance IL-10 production in a number of differenthuman and murine in vitro systems and murine disease models (Johnson etal., 2005, J Biol Chem 280:4037-4047 and International PatentApplication No. PCT/AU2005/000041, the disclosure of which isincorporated herein by reference), suggesting that Cpn10 hasconsiderable potential as an immune-therapeutic for the treatment ofautoimmune and inflammatory diseases.

However the site(s) within the Cpn10 molecule responsible for mediatingthis immunomodulatory activity(ies) has remained elusive.

The present invention relates to modifications to the Cpn10 moleculesand the effect of these modifications on immunomodulatory activity.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides Cpn10 polypeptidescomprising one or more amino acid substitutions, deletions and/oradditions compared to wild-type Cpn10, which polypeptides displayimmunomodulatory activity.

According to a first aspect of the present invention there is providedan isolated Cpn10 polypeptide possessing immunomodulatory activity butlacking, or substantially lacking, protein folding activity.

According to a second aspect of the present invention there is providedan isolated Cpn10 polypeptide having immunomodulatory activity, saidpolypeptide comprising one or more amino acid substitutions, deletionsand/or additions in the mobile loop region compared to a correspondingwild-type Cpn10 polypeptide.

The Cpn10 polypeptide may display immunomodulatory activity at leastsimilar to the level of immunomodulatory activity of the correspondingwild-type Cpn10 polypeptide.

In one embodiment, one or more residues of the IML tripeptide of themobile loop region of the Cpn10 polypeptide may be replaced with chargedresidues.

In another embodiment the Cpn10 polypeptide comprising the IMLtripeptide may be replaced with the tripeptide EEE. The Cpn10polypeptide comprising the EEE tripeptide may be set forth in SEQ ID NO:39. The Cpn10 polypeptide may be encoded by the nucleotide sequence asset forth in SEQ ID NO:40.

In yet another embodiment the Cpn10 polypeptide comprising the IMLtripeptide may be replaced with the tripeptide III. The Cpn10polypeptide comprising the III tripeptide may be set forth in SEQ ID NO:37. The Cpn10 polypeptide may be encoded by the nucleotide sequence asset forth in SEQ ID NO:38.

In still yet another embodiment the Cpn10 polypeptide comprising the IMLtripeptide may be replaced with the tripeptide IFI. The Cpn10polypeptide comprising the IFI tripeptide may be set forth in SEQ ID NO:35. The Cpn10 polypeptide may be encoded by the nucleotide sequence asset forth in SEQ ID NO:36.

According to a third aspect of the present invention there is providedan isolated Cpn10 polypeptide having immunomodulatory activity, saidpolypeptide substantially lacking the mobile loop region of acorresponding wild-type Cpn10 polypeptide.

In one embodiment the Cpn10 polypeptide substantially lacking the mobileloop region comprises the amino acid sequence as set forth in SEQ IDNO:3 or 24. The Cpn10 polypeptide substantially lacking the mobile loopregion may be encoded by the nucleotide sequence as set forth in SEQ IDNO:4, 5 or 25.

The Cpn10 polypeptide may display immunomodulatory activity at leastsimilar to the level of immunomodulatory activity of the correspondingwild-type Cpn10 polypeptide.

According to a fourth aspect of the present invention there is providedan isolated Cpn10 polypeptide having immunomodulatory activity, saidpolypeptide comprising one or more amino acid substitutions, deletionsand/or additions in the roof β-hairpin region compared to acorresponding wild-type Cpn10 polypeptide.

The Cpn10 polypeptide may display immunomodulatory activity at leastsimilar to the level of immunomodulatory activity of the correspondingwild-type Cpn10 polypeptide.

According to a fifth aspect of the present invention there is providedan isolated Cpn10 polypeptide having immunomodulatory activity, saidpolypeptide substantially lacking the roof β-hairpin region of acorresponding wild-type Cpn10 polypeptide.

In one embodiment the Cpn10 polypeptide substantially lacking the roofβ-hairpin region (SEQ ID NO:13) comprises the amino acid sequence as setforth in SEQ ID NO:6or 26. The Cpn10 polypeptide substantially lackingthe roof β-hairpin region may be encoded by the nucleotide sequence asset forth in SEQ ID NO:7, 8 or 27.

The Cpn10 polypeptide may display immunomodulatory activity at leastsimilar to the level of immunomodulatory activity of the correspondingwild-type Cpn10 polypeptide.

According to a sixth aspect of the present invention there is providedan isolated Cpn10 polypeptide having immunomodulatory activity, saidpolypeptide comprising one or more amino acid substitutions, deletionsand/or additions in the mobile loop region and the roof β-hairpin regioncompared to a corresponding wild-type Cpn10 polypeptide.

According to a seventh aspect of the present invention there is providedan isolated Cpn10 polypeptide, said polypeptide substantially lackingboth mobile loop region and the roof β-hairpin region of thecorresponding wild-type Cpn10 polypeptide.

The isolated Cpn10 polypeptide may display immunomodulatory activity atleast similar to the level of immunomodulatory activity of thecorresponding wild-type Cpn10 polypeptide.

In one embodiment the Cpn10 polypeptide comprises the amino acidsequence as set forth in SEQ ID NO:9 or 28. The Cpn10 polypeptide may beencoded by the nucleotide sequence as set forth in SEQ ID NO:10 or 29.

According to an eighth aspect of the present invention there is providedan isolated Cpn10 polypeptide having immunomodulatory activity, saidpolypeptide comprising a deletion of an extra N-terminal alanine residuecompared to a corresponding wild-type Cpn10 polypeptide.

The Cpn10 polypeptide may lack an acetyl group at the N terminuscompared to the corresponding wild-type Cpn10 polypeptide. The Cpn10polypeptide may display a reduced level of immunomodulatory activitywhen compared to the level of immunomodulatory activity of thecorresponding wild-type Cpn10 polypeptide.

In one embodiment the Cpn10 polypeptide comprising the deletion of theextra N-terminal alanine residue may comprise the amino acid sequence asset forth in SEQ ID NO:23. The Cpn10 polypeptide comprising the deletionof the extra N-terminal alanine residue may be encoded by the nucleotidesequence as set forth in SEQ ID NO:44.

According to a ninth aspect of the present invention there is providedan isolated Cpn10 polypeptide having immunomodulatory activity, whereinthe N terminus of the Cpn10 polypeptide is substantially replaced with abacterial Cpn10 N terminus.

The bacterial Cpn10 may be GroES.

The Cpn10 polypeptide may display a reduced level of immunomodulatoryactivity when compared to the level of immunomodulatory activity of thecorresponding wild-type Cpn10 polypeptide.

In one embodiment the Cpn10 polypeptide may comprise the amino acidsequence as set forth in SEQ ID NO:14. The Cpn10 polypeptide may beencoded by the nucleotide sequence as set forth in SEQ ID NO:43.

According to a tenth aspect of the present invention there is providedan isolated Cpn10 polypeptide having immunomodulatory activity, whereina glycine residue replaces an extra N terminal alanine residue of theCpn10 polypeptide as compared to a corresponding wild-type Cpn10polypeptide.

The Cpn10 polypeptide may display a reduced level of immunomodulatoryactivity when compared to the level of immunomodulatory activity of thecorresponding wild-type Cpn10 polypeptide.

In one embodiment the Cpn10 polypeptide may comprise the amino acidsequence as set forth in SEQ ID NO:30. The Cpn10 polypeptide may beencoded by the nucleotide sequence as set forth in SEQ ID NO:31.

According to an eleventh aspect of the present invention there isprovided an isolated nucleic acid encoding a Cpn10 polypeptide accordingto any one of the first to the tenth aspects.

According to a twelfth aspect of the present invention there is providedan expression construct comprising a nucleic acid according to theeleventh aspect operably-linked to one or more regulatory sequences.

According to a thirteenth aspect of the present invention there isprovided a host cell expressing a polypeptide of any one of the first totenth aspects, or comprising a nucleic acid of the eleventh aspect or anexpression construct of the ninth aspect.

According to a fourteenth aspect of the present invention there isprovided an antibody that selectively binds to a polypeptide of any oneof the first to the tenth aspects.

According to a fifteenth aspect of the present invention there isprovided a pharmaceutical composition comprising a polypeptide of anyone of the first to the tenth aspects, a nucleic add of the eleventh, anexpression construct of the twelfth aspect or an antibody of thefourteenth aspect.

The pharmaceutical composition may comprise one or more additionalagents. For example, for the treatment of multiple sclerosis thecomposition may further comprise an effective amount of IFNβ.

According to a sixteenth aspect of the present Invention there isprovided a method of treating a subject, including the step ofadministering to said subject an effective amount of a Cpn10 polypeptideof any one of the first to the tenth aspects or a nucleic acid of theeleventh aspect.

The treatment may modulate the immune response in the subject. Theimmune response may be modulated via modulation of Toll-like receptorsignaling.

According to a seventeenth aspect of the present invention there isprovided a method for treating or preventing a disease or condition in asubject, the method comprising administering to the subject an effectiveamount of a Cpn10 polypeptide of any one of the first to the tenthaspects or a nucleic acid of the eleventh aspect.

The disease, disorder or condition may be selected from acute or chronicinflammatory diseases, asthma, allergy, multiple sclerosis, GVHD, or aninfectious disease. The infectious disease may result from a bacterialor viral infection. The bacteria may be a Gram negative bacteria.

According to a eighteenth aspect of the present invention there isprovided a method for modulating TLR4 signalling in a subject, or in atleast one cell, tissue or organ thereof, the method comprisingadministering an effective amount of a Cpn10 polypeptide of any one ofthe first to the tenth aspects or a nucleic acid of the eleventh aspect.

Typically the Cpn10 regulates agonist-induced TLR4 signalling.

According to a nineteenth aspect of the present invention there isprovided a method for modulating the production and/or secretion of oneor more immunomodulators in a subject, or at least one cell, tissue ororgan thereof, the method comprising administering an effective amountof a Cpn10 polypeptide of any one of the first to the tenth aspects or anucleic acid of the eleventh aspect.

The Cpn10 may modulate signalling from TLR4.

The immunomodulator may be a pro-inflammatory cytokine or chemokine oran anti-inflammatory cytokine or chemokine. The cytokine or chemokinemay be selected from TNF-α, IL-6, RANTES, IL-10, TGF-β or a type Iinterferon. The type I interferon may be IFNα or IFNβ.

According to a twentieth aspect of the present invention there isprovided a method of identifying a compound that binds to a polypeptideof any one of the first to the tenth aspects, the method comprising thesteps of:

(a) contacting a candidate compound with said polypeptide; and

(b) assaying for the formation of a complex between the candidatecompound and said polypeptide.

The assay for the formation of a complex may be a competitive bindingassay or a two-hybrid assay.

According to a twenty-first aspect of the present invention there isprovided a method of screening for a compound that modulates theactivity of a polypeptide of any one of the first to the tenth aspect,the method comprising the steps of:

(a) contacting said polypeptide with a candidate compound underconditions suitable to enable interaction of said candidate compound tosaid polypeptide; and

(b) assaying for activity of said polypeptide.

Assaying for activity of the polypeptide may comprise adding a labeledsubstrate and measuring a change in the labeled substrate.

The invention also contemplates variants, derivatives, homologues,analogues and fragments of the modified Cpn10 polypeptides andpolynucleotides according to the above aspects and embodiments.

According to the above aspects and embodiments the Cpn10 polypeptidesand polynucleotides may be derived from any animal, may be generatedusing recombinant DNA technologies or may be synthetically produced.Typically the Cpn10 is a eukaryotic Cpn10.

According to the above aspects the wild type Cpn10 polypeptide may be ahuman Cpn10 polypeptide comprising the amino acid sequence as set forthin SEQ ID NO:1 or 21.

According to the above aspects the wild type Cpn10 polypeptide may beencoded by the nucleotide sequence as set forth in SEQ ID NO:2 or 22.

According to the above aspects and embodiments the immunomodulatoryactivity of a Cpn10 polypeptide may involve generation of heptamers ofthe polypeptide.

DEFINITIONS

In the context of this specification, the term “comprising” means“including principally, but not necessarily solely”. Furthermore,variations of the word “comprising”, such as “comprise” and “comprises”,have correspondingly varied meanings.

The term “wild-type” as used herein in relation to Cpn10 polypeptidesincludes polypeptides in their native or non-native form. For example,native human Cpn10 is acetylated at its N-terminus; the presentinvention contemplates, within the scope of the term wild-type,polypeptides acetylated or non-acetylated. Further, wild type Cpn10polypeptides may comprise an additional alanine (A) reside at theN-terminus (WO 2004/041300, the disclosure of which is incorporatedherein by reference).

The term “polypeptide” means a polymer made up of amino acids linkedtogether by peptide bonds. The terms “polypeptide” and “protein” areused interchangeably herein, although for the purposes of the presentinvention a “polypeptide” may constitute a portion of a full lengthprotein.

The term “polynucleotide” as used herein refers to a single- ordouble-stranded polymer of deoxyribonucleotide, ribonucleotide bases orknown analogues of natural nucleotides, or mixtures thereof. The termincludes reference to the specified sequence as well as to the sequencecomplimentary thereto, unless otherwise indicated. The terms“polynucleotide” and “nucleic acid” are used interchangeably herein.

The term “isolated” means that the molecule in question has been removedfrom its natural environment or host, and associated impurities reducedor eliminated such that the molecule in question is the predominantspecies present (ie., on a molar basis it is more abundant than anyother individual species in the composition/sample). Typically asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 30 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 to 90 percent of allmacromolecular species present in the composition. Most typically, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies.

As used herein the term “substantially” means the majority but notnecessarily all, and thus in relation to a modified polypeptide“substantially” lacking a component region of a corresponding wild-typepolypeptide, the modified polypeptide may retain a portion of thatcomponent region. For example, a modified polypeptide “substantially”lacking a component region of a corresponding wild-type polypeptide mayretain, approximately 50 percent or less of the sequence of thecomponent region, although typically the component region is renderedstructurally and/or functionally inactive by virtue of the proportion ofthe sequences of the region omitted.

The term “conservative amino acid substitution” as used herein refers toa substitution or replacement of one amino acid for another amino acidwith similar properties within a polypeptide chain (primary sequence ofa protein). For example, the substitution of the charged amino acidglutamic acid (Glu) for the similarly charged amino acid aspartic acid(Asp) would be a conservative amino acid substitution.

As used herein the terms “treatment”, “treating” and variations thereof,refer to any and all uses which remedy a disease state or symptoms,prevent the establishment of disease, or otherwise prevent, hinder,retard, or reverse the progression of disease or other undesirablesymptoms in any way whatsoever.

As used herein the term “effective amount” includes within its meaning anon-toxic but sufficient amount of an agent or compound to provide thedesired therapeutic or prophylactic effect. The exact amount requiredwill vary from subject to subject depending on factors such as thespecies being treated, the age and general condition of the subject, theseverity of the condition being treated, the particular agent beingadministered and the mode of administration and so forth. Thus, it isnot possible to specify an exact “effective amount”. However, for anygiven case, an appropriate “effective amount” may be determined by oneof ordinary skill in the art using only routine experimentation.

As used herein the terms “modulating”, “modulates” and variationsthereof refer to increasing or decreasing the level of activity,production, secretion or functioning of a molecule in the presence of aparticular molecule or agent of the invention compared to the level ofactivity, production, secretion or other functioning thereof in theabsence of the molecule or agent. These terms do not implyquantification of the increase or decrease. The modulation may be of anymagnitude sufficient to produce the desired result and may be direct orindirect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A. Crystal structure of E. coli Cpn10 (GroES) showing theanti-parallel β-barrel, the “roof” β-hairpin loop and mobile loopregions. Cpn10 is comprised of seven identical 10 kDa subunits. B. Aminoacid sequence of wild-type human X-Cpn10 monomer (SEQ ID NO:23).Predicted 18 amino acid mobile loop in bold, italic. Predicted 14 aminoacid roof β-hairpin in bold, underline.

FIG. 2. Effect of Human Ala-Cpn10 and E. coli GroES on TLR4 signaling.Dose-responsive inhibition of LPS-induced HIV-LTR activation (anindirect measure of NFκB activity) by human Ala-Cpn10 (batch CH001) butnot E. coli GroES. Panel B show the results from panel A as percentinhibition of luciferase activity (NFκB activity) relative to the levelsof luciferase produced with LPS alone. The LPS alone samples are themean of 4 replicates, all other samples are the mean of 2 replicates.RLU=relative light units. NFκB activity was induced with 5 ng/mlLipopolysaccharide (LPS).

FIG. 3. SDS-PAGE gels. Lane assignments for gels A to O, except H: Lane1, molecular weight markers (kDa); lanes 2 to 6, 60 μg, 6 μg, 3 μg, 1.2μg and 0.3 μg of Cpn10 respectively A. 4-12% SDS-PAGE gel stained withCoomassie brilliant blue of purified Ala-Cpn10 (CH001); B. 4-12%SDS-PAGE gel stained with Coomassie brilliant blue of purified Ala-Cpn10(CH003); C. 4-12% SDS-PAGE gel stained with Coomassie brilliant blue ofpurified Ala-Cpn10-EEE-cHis; D. 4-12% SDS-PAGE gel stained withCoomassie brilliant blue of purified Ala-Cpn10-cHis; E. 4-12% SDS-PAGEgel stained with Coomassie brilliant blue of purified Ala-Cpn10-IFI; F.4-12% SDS-PAGE gel stained with Coomassie brilliant blue of purifiedAla-Cpn10-III; G. 4-12% SDS-PAGE gel stained with Coomassie brilliantblue of purified Ala-Cpn10-Δml; H. Partial glutaraldehyde cross-linkingof Cpn10-Δml (lane 2) shows 7 distinct bands on silver stained 4-12%SDS-PAGE gel, revealing the heptameric structure of the molecule. Lane1, molecular weight markers (kDa). I. 4-12% SDS-PAGE gel stained withCoomassie brilliant blue of purified Ala-Cpn10-Δroof. J. 4-12% SDS-PAGEgel stained with Coomassie brilliant blue of purifiedAla-Cpn10-β-barrel. K. 4-12% SDS-PAGE gel stained with Coomassiebrilliant blue of purified E. coli GroES; L. 4-12% SDS-PAGE gel stainedwith Coomassie brilliant blue of purified Cpn10-NtermES; M. 4-12%SDS-PAGE gel stained with Coomassie brilliant blue of purified E. coliGroES; N: 4-12% SDS-PAGE gel stained with Coomassie brilliant blue ofpurified Gly-Cpn10.

FIG. 4. Effect of Ala-Cpn10, Ala-Cpn10-III, Ala-Cpn10-IFI,Ala-Cpn10-EEE-cHis and Ala-Cpn10-cHis on TLR4 signalling.Dose-responsive inhibition of LPS-induced HIV-LTR activation by humanAla-Cpn10 (batch CH001), Ala-Cpn10 C-terminal hexahistidine tag(Ala-Cpn10-cHis) and numerous mobile loop mutants, Panels B,D,F,H showthe results from panels A,C,E,G as percent inhibition of luciferaseactivity (NFκB activity) relative to the levels of luciferase producedwith LPS alone. The LPS alone samples are the mean of 4 replicates, allother samples are the mean of 2 replicates. RLU=relative light units.NFκB activity was induced with 5 ng/ml Lipopolysaccharide (LPS).

FIG. 5. Effect of Ala-Cpn10 and Ala-Cpn10Δml on TLR4 signalling.Dose-responsive inhibition of LPS-induced HIV-LTR activation by humanAla-Cpn10 (batch CH001) and Ala-Cpn10-Δml. Panel B shows the resultsfrom panel A as percent inhibition of luciferase activity (NFκBactivity) relative to the levels of luciferase produced with LPS alone.The LPS alone samples are the mean of 4 replicates, all other samplesare the mean of 2 replicates. RLU=relative light units. NFκB activitywas induced with 5 ng/ml Lipopolysaccharide (LPS).

FIG. 6. Effect of Ala-Cpn10 and Ala-Cpn10Δroof and E. coli GroES on TLR4signaling, Dose-responsive inhibition of LPS-induced HIV-LTR activationby human Ala-Cpn10 (batch CH001) and Ala-Cpn10-Δroof. Panel B shows theresults from panel A as percent inhibition of luciferase activity (NFκBactivity) relative to the levels of luciferase produced with LPS alone.The LPS alone samples are the mean of 6 replicates, all other samplesare the mean of 2 replicates. CPS=relative counts per seconds. NFκBactivity was induced with 5 ng/ml Lipopolysaccharide (LPS).

FIG. 7. Effect of Ala-Cpn10 and Ala-Cpn10-β-barrel on TLR4 signalling.Dose-responsive inhibition of LPS-induced HIV-LTR activation by humanAla-Cpn10 (batch CH001) and Ala-Cpn10-β-barrel. Panel B shows theresults from panel A as percent inhibition of luciferase activity (NFκBactivity) relative to the levels of luciferase produced with LPS alone.The LPS alone samples are the mean of 6 replicates, all other samplesare the mean of 2 replicates. CPS=relative counts per seconds. NFκBactivity was induced with 5 ng/ml Lipopolysaccharide (LPS).

FIG. 8. Effect of Ala-Cpn10 and Cpn10-NtermES on TLR4 signalling.Dose-responsive inhibition of LPS-induced HIV-LTR activation by humanwild-type Cpn10 (batch CH001) but not Cpn10-NtermES. Panel B shows theresults from panel A as percent inhibition of luciferase activity (NFκBactivity) relative to the levels of luciferase produced with LPS alone.The LPS alone samples are the mean of 6 replicates, all other samplesare the mean of 2 replicates. CPS=relative counts per seconds;SD=standard deviation. NFκB activity was induced with 5 ng/mlLipopolysaccharide (LPS).

FIG. 9. Effect of Ala-Cpn10 and X-Cpn10 on TLR4 signalling.Dose-responsive inhibition of LPS-induced HIV-LTR activation by humanAla-Cpn10 (batch CH003) and X-Cpn10. Panel B shows the results frompanel A as percent inhibition of luciferase activity (NFκB activity)relative to the levels of luciferase produced with LPS alone. The LPSalone samples are the mean of 6 replicates, all other samples are themean of 2 replicates. CPS=relative counts per seconds. NFκB activity wasinduced with 5 ng/ml Lipopolysaccharide (LPS).

FIG. 10. Effect of Ala-Cpn10 and Gly-Cpn10 on TLR4 signalling.Dose-responsive inhibition of LPS-induced HIV-LTR activation by humanAla-Cpn10 (batch CH003) and Gly-Cpn10. Panel B shows the results frompanel A as percent inhibition of luciferase activity (NFκB activity)relative to the levels of luciferase produced with LPS alone. The LPSalone samples are the mean of 6 replicates, all other samples are themean of 2 replicates. CPS=relative counts per seconds. NFκB activity wasinduced with 5 ng/ml Lipopolysaccharide (LPS).

FIG. 11. Effect of Ala-Cpn10 and Ala-Cpn10-Δml on TLR4 signalling.Dose-responsive inhibition of LPS-induced HIV-LTR activation by humanAla-Cpn10 (batch CH003) and Ala-Cpn10-Δml. Panel B shows the resultsfrom panel A as percent inhibition of luciferase activity (NFκBactivity) relative to the levels of luciferase produced with LPS alone.The LPS alone samples are the mean of 6 replicates, all other samplesare the mean of 2 replicates. CPS=relative counts per seconds. NFκBactivity was induced with 5 ng/ml Ultra-Pure Lipopolysaccharide (LPS).

FIG. 12. Effect of Ala-Cpn10 and Ala-Cpn10-Δroof on TLR4 signalling,Dose-responsive inhibition of LPS-induced HIV-LTR activation by humanAla-Cpn10 (batch CH003) and Ala-Cpn10-Δroof. Panel B shows the resultsfrom panel A as percent inhibition of luciferase activity (NFκBactivity) relative to the levels of luciferase produced with LPS alone.The LPS alone samples are the mean of 6 replicates, all other samplesare the mean of 2 replicates. CPS=relative counts per seconds, NFκBactivity was induced with 5 ng/ml Ultra-Pure Lipopolysaccharide (LPS).

FIG. 13. Effect of Ala-Cpn10 and Ala-Cpn10-β-barrel on TLR4 signalling.Dose-responsive inhibition of LPS-induced HIV-LTR activation by humanAla-Cpn10 (batch CH003) and Ala-Cpn10-β-barrel. Panel B shows theresults from panel A as percent inhibition of luciferase activity (NFκBactivity) relative to the levels of luciferase produced with LPS alone.The LPS alone samples are the mean of 6 replicates, all other samplesare the mean of 2 replicates, CPS=relative counts per seconds. NFκBactivity was induced with 5 ng/ml Ultra-Pure Lipopolysaccharide (LPS).

FIG. 14. Effect of Ala-Cpn10 and Gly-Cpn10 on TLR4 signalling,Dose-responsive inhibition of LPS-induced HIV-LTR activation by humanAla-Cpn10 (batch CH003) and Gly-Cpn10. Panel B shows the results frompanel A as percent inhibition of luciferase activity (NFκB activity)relative to the levels of luciferase produced with LPS alone. The LPSalone samples are the mean of 6 replicates, all other samples are themean of 2 replicates. CPS=relative counts per seconds. NFκB activity wasinduced with 5 ng/ml Ultra-Pure Lipopolysaccharide (LPS).

FIG. 15. Cpn10 activity in a murine inflammatory model of endotoxaemia.Cpn10 and Cpn10 variants reduced LPS-induced serum TNF-α, IL-10 and IL-6production. Sera from ‘LPS challenged’ (see Table 1, groups 1, 3, 5, 7,9 and 11) or ‘saline control’ (see Table 1, groups 2, 4, 6, 8, 10 and12) mice were analyzed for inflammatory-associated cytokines using CBA(see Methods). The levels of A, B) TNF-α, C, D) IL-6, and E, F) IL-10cytokines were plotted with the mean of each group displayed (horizontalbar). 1-way ANOVA analysis with Tukeys post-hoc test was performed foreach data set. Statistical significance in the data is indicated inbrackets (p<0.05) (see text for details).

FIG. 16 Diagram of the N terminus of Acetyl-Cpn10, Ala-Cpn10 andGly-Cpn10.

SEQUENCE REFERENCES

Table 1. The reference table below provides nomenclature of Cpn10polypeptides used throughout this specification. This table containsdescriptions of features of Cpn10 polypeptides disclosed herein andtheir corresponding amino acid and nucleic acid assigned sequenceidentifiers.

TABLE 1 The reference table below provides nomenclature of Cpn10polypeptides used throughout this specification. This table containsdescriptions of features of Cpn10 polypeptides disclosed herein andtheir corresponding amino acid and nucleic acid assigned sequenceidentifiers. Amino Nucleic Acid Acid SEQ ID SEQ ID Cpn10 name FeatureAdditional Feature NO. NO. X-Cpn10 Does not include an Does not include23 44 (i.e. non- acetyl group at the N initiating Methionine acetylated)terminus Cpn10 (Wild type) Does not include an Includes initiating 1  2acetyl group Methionine Cpn10-Δml Mobile loop deleted Includesinitiating 3 4, 5 Methionine Cpn10-Δroof Roof loop deleted Includesinitiating 6 7, 8 Methionine Cpn10 β-barrel Roof loop and mobileIncludes initiating 9 10 loop deleted Methionine E. coli Cpn10 Bacterialhomolog of Includes initiating 11 34 (GroES) Cpn10 Methionine Mobileloop only Example of a mobile 12 loop sequence in human Cpn10 Betahairpin roof Example of a roof loop 13 (“roof”) loop only sequence inhuman Cpn10 Cpn10-NtermES Human Cpn10 except Includes initiating 14 43for E. coli N terminus Methionine Forward primer EEE tripeptide is 15for EEE located in the mobile tripeptide loop Reverse primer EEEtripeptide is 16 for EEE located in the mobile tripeptide loop Forwardprimer IFI tripeptide is located 17 for generating in the mobile loopIFI tripeptide Reverse primer IFI tripeptide is located 18 forgenerating in the mobile loop IFI tripeptide Forward primer IIItripeptide is located 19 for generating III in the mobile looptripeptide Reverse primer III tripeptide is located 20 for IIItripeptide in the mobile loop Ala-Cpn10 (Wild Extra N terminal Does notinclude 21 22 type Cpn10) alanine residue initiating MethionineAla-Cpn10-Δml Mobile loop deleted Does not include 24 25 initiatingMethionine but includes extra N terminal alanine residue Ala-Cpn10- Roofloop deleted Does not include 26 27 Δroof initiating Methionine butincludes extra N terminal alanine residue Ala-Cpn10 β- Roof loop andmobile Does not include 28 29 barrel loop deleted initiating Methioninebut includes extra N terminal alanine residue Gly-Cpn10 Glycine replacesthe 30 31 extra N terminal alanine residue Ala-Cpn10-IFI IFI tripeptidereplaces Extra N terminal 35 36 IML tripeptide in mobile alanine residueloop Ala-Cpn10-III III tripeptide replaces Extra N terminal 37 38 IMLtripeptide in mobile alanine residue loop Ala-Cpn10- EEE tripeptideExtra N terminal 39 40 EEE-cHis replaces IML tripeptide alanine residueand in mobile loop a His tag at the C terminus Ala-Cpn10-cHis Extra Nterminal His tag at the C 41 42 alanine residue terminus

BEST MODE OF PERFORMING THE INVENTION

Cpn10 is a dome-shaped, heptameric ring of identical 10 kDa subunits(see FIG. 1), The inner surface of the dome is hydrophilic and highlycharged. Each Cpn10 subunit forms an irregular β-barrel topology fromwhich two large extensions protrude. The first extension is a β-hairpinloop that extends towards the center of the heptamer and forms thedome-like structure. Intriguingly, whereas the roof of GroES (E. ColiCpn10) is charged negatively under physiological conditions, the roof ofmammalian Cpn10 is positively charged; while a large portion of the roofis missing completely from the bacteriophage Cpn10 (Gp31). The moleculealso has another extension that is a flexible 18 amino acid mobile loopthat extends from the base of the dome and mediates an interaction withCpn60. Site-directed mutagenesis has identified several residues withinthe mobile loop which are crucial for the interaction with Cpn60, namelythree hydrophobic residues (30-IML-32) at the base of the mobile loopwhich constitute the actual Cpn60-binding site and two residues (26-Tand 33-P) which restrict the flexibility of the mobile loop (Richardsonet al., 2001, J Biol Chem 276:4981-4987). Therefore, the association ofCpn10 with Cpn60 is mediated by the 18 amino acid mobile loop of Cpn10(see FIG. 1D). In E. coli GroES the Cpn60/GroEL-binding site tripeptideis less hydrophobic (25-IVL-27) and the mobile loop is more flexiblethan mammalian Cpn10. These changes decrease the affinity of GroES forCpn60/GroEL and as a result, GroES can not form a productive interactionwith Cpn60 white both Cpn10 and GroES function with GroEL.

Beginning with the hypothesis that the mechanism by which extracellularCpn10 produces its immunomodulatory effects involves Cpn60 (Johnson etal., 2005, J Biol Chem 280:4037-4047), the present inventors generatedsite-specific mutants of Cpn10 targeting the mobile loop region anddemonstrate herein that mutations which perturb interactions with Cpn60retain immunomodulatory activity.

Accordingly, in one aspect the present invention provides isolated Cpn10polypeptides displaying immunomodulatory activity but substantiallylacking protein folding ability.

It is also demonstrated herein that deletion of a substantial portion ofthe mobile loop region and/or the roof β-hairpin region of Cpn10 doesnot abolish the ability of Cpn10 to modulate signalling from theToll-like receptor TLR4.

Accordingly, the present invention also provides isolated Cpn10polypeptides having immunomodulatory activity, the polypeptidescomprising one or more amino acid substitutions, deletions and/oradditions in one or both of the mobile loop region and the roofβ-hairpin region compared to a corresponding wild-type Cpn10polypeptide. The deletion of the mobile loop and the roof loop of Cpn10is termed the Ala-Cpn10-β-barrel polypeptide as disclosed herein.

The present invention also provides isolated Cpn10 polypeptidessubstantially lacking one or both of the mobile loop region and the roofβ-hairpin region of the corresponding wild-type Cpn10 polypeptide.

As described herein the present inventors have also demonstrated that E.coli GroES is not capable of inducing the immunomodulatory effectattributable to human Cpn10, as determined by modulation of TLRsignalling. Further, the inactivity of a Cpn10 polypeptide in which theN-terminal residues of human Cpn10 have been replaced by thecorresponding N-terminal residues from E. coli GroES demonstrates thatthe N-terminus of Cpn10 is required for immunomodulatory activity.

As further described herein the present inventors have also demonstratedthat the addition of a glycine residue to the N terminus of Cpn10augments immunomodulatory activity. It is contemplated that the presenceof an acetyl group or an amino acid which shares structural homology toan acetyl group such as an alanine residue or a glycine residue augmentsimmunomodulatory activity of Cpn10.

Polypeptides

As disclosed herein the present invention contemplates Cpn10polypeptides, typically possessing immunodulatory activity, comprisingone or more amino acid deletions, additions or substitutions incomparison with a corresponding wild-type Cpn10 polypeptide. Typicallythe wild-type Cpn10 is any Cpn10 polypeptide from a eukaryotic organism.By way of example, the Cpn10 may be derived from yeast (e.g.Saccharomyces cerevisiae), nematode (e.g. Caenorhabditis elegans), frog(e.g. Xenopus tropicalis), chicken (e.g. Gallus gallus), zebrafish (e.g.Danio rerio), fly (e.g. fruit fly such as Drosphila melanogaster), plant(e.g. Arabidopsis thaliana) or a mammal. The mammalian Cpn10 may beprimate, murine, ovine, bovine, canine, feline, porcine or equine.Alternatively the Cpn10 may be archaeal in origin. In particularembodiments the Cpn10 is human Cpn10. The amino acid sequence of thewild-type human Cpn10 may be as set forth in SEQ ID NO:1 or 21. Thenucleotide sequence encoding the wild-type Cpn10 may be as set forth inSEQ ID NO:2 or 22 or display sufficient sequence identity thereto tohybridize to the sequence of SEQ ID NO:2 or 22.

The present invention relates to modifications of wild-type Cpn10polypeptides as disclosed herein and encompasses otherwise wild-typemolecules modified at the N-terminus or C-terminus by the addition,deletion, or substitution of one or more amino acid residues. Forexample, amino acid additions may result from the fusion of a Cpn10polypeptide or fragment thereof with a second polypeptide or peptide,such as a polyhistidine tag, maltose binding protein fusion, glutathioneS transferase fusion, green fluorescent protein fusion, or the additionof an epitope tag such as FLAG or c-myc. For example, a modification ofa wild-type human Cpn10 polypeptide may comprise an additional glycine(G) residue. The Cpn10 polypeptide may or may not include the initiatingmethionine at the N terminus.

In the case of immunomodulatory Cpn10 polypeptides of the inventionbased on, or substantially derived from human Cpn10, such polypeptidestypically comprise the N-terminal sequence MAGQAFRKFL (SEQ ID NO:32),optionally including one or more modifications as described above.

As disclosed herein, Cpn10 polypeptides of the invention may compriseone or more amino acid additions, deletions or substitutions in eitheror both of the mobile loop region and the roof β-hairpin region. In oneembodiment, one or more amino acid substitutions may be made in themobile loop region, for example within the tripeptide sequenceresponsible for interaction with Cpn60, such that the modifiedpolypeptide retains immunomodulatory activity but does not retainprotein folding activity. In an alternative embodiment the Cpn10polypeptide substantially lacks the mobile loop region, for example asexemplified by the sequence set forth in SEQ ID NO:3 or 24, or the roofβ-hairpin region, for example as exemplified by the sequence set forthin SEQ ID NO:6 or 26, or both the mobile loop and roof β-hairpinregions, for example as exemplified by the sequence set forth in SEQ IDNO:9 or 28.

As defined herein, the amino acids constituting the mobile loop or roofβ-hairpin are defined on the basis of the sequence and known crystalstructure of the E. coli Cpn10, GroES. The locations of the mobile loopand roof β-hairpin regions in eukaryotic Cpn10 polypeptides arepredicted to be similar in view of the conservation of Cpn10 sequencesthrough evolution and the conservation of predicted three-dimensionalprotein structures. However the precise boundaries of the mobile loopand roof β-hairpin regions in eukaryotic Cpn10 polypeptides may differslightly from those of GroES.

The term “variant” as used herein refers to substantially similarsequences. Generally, polypeptide sequence variants possess qualitativebiological activity in common. Further, these polypeptide sequencevariants may share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% sequence identity. Also included within themeaning of the term “variant” are homologues of polypeptides of theinvention. A homologue is typically a polypeptide from a differentspecies but sharing substantially the same biological function oractivity as the corresponding polypeptide disclosed herein.

Further, the term “variant” also includes analogues of the polypeptidesof the invention, wherein the term “analogue” means a polypeptide whichis a derivative of a polypeptide of the invention, which derivativecomprises addition, deletion, substitution of one or more amino acids,such that the polypeptide retains substantially the same function. Theterm “conservative amino acid substitution” refers to a substitution orreplacement of one amino acid for another amino acid with similarproperties within a polypeptide chain (primary sequence of a protein).

The present invention also contemplates fragments of the polypeptidesdisclosed herein. The term “fragment” refers to a polypeptide moleculethat encodes a constituent or is a constituent of a polypeptide of theinvention or variant thereof. Typically the fragment possessesqualitative biological activity in common with the polypeptide of whichit is a constituent. The peptide fragment may be between about 5 toabout 150 amino acids in length, between about 5 to about 100 aminoacids in length, between about 5 to about 50 amino acids in length, orbetween about 5 to about 25 amino acids in length. Alternatively, thepeptide fragment may be between about 5 to about 15 amino acids inlength.

Cpn10 polypeptides modified at the N- and/or C-terminus by the addition,deletion or substitution of one or more amino acid residues as describedabove also fall within the scope of the present invention.

Production of Cpn10

In accordance with the present invention Cpn10 polypeptides may beproduced using standard techniques of recombinant DNA and molecularbiology that are well known to those skilled in the art. Guidance may beobtained, for example, from standard texts such as Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York,1989 and Ausubel et al., Current Protocols in Molecular Biology, GreenePubl. Assoc. and Wiley-Intersciences, 1992. Methods described in Mortonet al., 2000 (Immunol Cell Biol 78:603-607), Ryan et al., 1995 (J BiolChem 270:22037-22043) and Johnson et al., 2005 (J Biol Chem280:4037-4047) are examples of suitable purification methods for Cpn10polypeptides, although the skilled addressee will appreciate that thepresent invention is not limited by the method of purification orproduction used and any other method may be used to produce Cpn10 foruse in accordance with the methods and compositions of the presentinvention. Cpn10 peptides may be produced by digestion of a polypeptidewith one or more proteinases such as endoLys-C, endoArg-C, endoGlu-C andstaphylococcus V8-protease. The digested peptide fragments can bepurified by, for example, high performance liquid chromatographic (HPLC)techniques.

The purification of Cpn10 polypeptides of the invention may be scaled-upfor large-scale production purposes. For example, as described hereinthe present inventors have developed a bioprocess for the production oflarge (gram) quantities of highly pure, clinical grade Cpn10polypeptides by batch fermentation in E. coli.

Cpn10 polypeptides of the present invention, as well as fragments andvariants thereof, may also be synthesised by standard methods of liquidor solid phase chemistry well known to those of ordinary skill in theart. For example such molecules may be synthesised following the solidphase chemistry procedures of Steward and Young (Steward, J. M. & Young,J. D., Solid Phase Peptide Synthesis. (2nd Edn.) Pierce Chemical Co.,Illinois, USA (1984).

In general, such a synthesis method comprises the sequential addition ofone or more amino acids or suitably protected amino acids to a growingpeptide chain. Typically, either the amino or carboxyl group of thefirst amino acid is protected by a suitable protecting group. Theprotected amino acid is then either attached to an inert solid supportor utilised in solution by adding the next amino acid in the sequencehaving the complimentary (amino or carboxyl) group suitably protectedand under conditions suitable for forming the amide linkage. Theprotecting group is then removed from this newly added amino acidresidue and the next (protected) amino acid is added, and so forth.After all the desired amino acids have been linked, any remainingprotecting groups, and if necessary any solid support, is removedsequentially or concurrently to produce the final polypeptide.

Amino acid changes in Cpn10 polypeptides may be effected by techniqueswell known to those persons skilled in the relevant art. For example,amino acid changes may be effected by nucleotide replacement techniqueswhich include the addition, deletion or substitution of nucleotides(conservative and/or non-conservative), under the proviso that theproper reading frame is maintained. Exemplary techniques include randommutagenesis, site-directed mutagenesis, oligonucleotide-mediated orpolynucleotide-mediated mutagenesis, deletion of selected region(s)through the use of existing or engineered restriction enzyme sites, andthe polymerase chain reaction.

The generation of immunomodulatory activity by the Cpn10 polypeptides ofthe invention may involve the formation of heptamers of the Cpn10polypeptides. Testing of immunomodulatory activity for the purposes ofthe present invention may be via any one of a number of techniques knownto those of skill in the art. As exemplified herein immunomodulatoryactivity of Cpn10 polypeptides may be determined by measuring theability of the polypeptide to modulate signalling from the Toll-likereceptor TLR4, for example using a luciferase bioassay, and typically inthe presence of a TLR4 agonist such as lipopolysaccharide. Alternativelyor in addition, immunomodulatory activity may be determined using otherassays in vitro, ex vivo or in vivo, for example via measurement ofNF-κB production or the production of cytokines in cells such asperipheral blood mononuclear cells.

Polynucleotides

Embodiments of the present invention provide isolated polynucleotidesencoding Cpn10 polypeptides as described above, and variants andfragments of such polynucleotides. The nucleotide sequences encodingwild-type Cpn10 may be as set forth in SEQ ID NO:2 or 22 or displaysufficient sequence identity thereto to hybridize to the sequence of SEQID NO:2 or 22.

Specifically, the nucleotide sequence encoding the Cpn10-NtermESpolypeptide of the invention may be as set forth in SEQ ID NO:43 ordisplay sufficient sequence identity thereto to hybridize to thesequence of SEQ ID NO:43.

The nucleotide sequence encoding the Ala-Cpn10 polypeptide of theinvention may be as set forth in SEQ ID NO:22 or display sufficientsequence identity thereto to hybridize to the sequence of SEQ ID NO:22.

The nucleotide sequence encoding the Ala-Cpn10-Δml polypeptide of theinvention may be as set forth in SEQ ID NO:25 or display sufficientsequence identity thereto to hybridize to the sequence of SEQ ID NO:25.

The nucleotide sequence encoding the Ala-Cpn10-Δroof polypeptide of theinvention may be as set forth in SEQ ID NO:27 or display sufficientsequence identity thereto to hybridize to the sequence of SEQ ID NO:27.

The nucleotide sequence encoding the Cpn10 β-barrel polypeptide of theinvention may be as set forth in SEQ ID NO:10 or display sufficientsequence identity thereto to hybridize to the sequence of SEQ ID NO:10.The nucleotide sequence encoding the Ala-Cpn10 β-barrel polypeptide ofthe invention may be as set forth in SEQ ID NO:29 or display sufficientsequence identify thereto to hybridize to the sequence of SEQ ID NO:29.

The nucleotide sequence encoding the Gly-Cpn10 polypeptide of theinvention may be as set forth in SEQ ID NO:31 or display sufficientsequence identity thereto to hybridize to the sequence of SEQ ID NO:31.

The nucleotide sequence encoding the Ala-Cpn10-IFI polypeptide of theinvention may be as set forth in SEQ ID NO:36 or display sufficientsequence identity thereto to hybridize to the sequence of SEQ ID NO:36.

The nucleotide sequence encoding the Ala-Cpn10-III polypeptide of theinvention may be as set forth in SEQ ID NO:38 or display sufficientsequence identity thereto to hybridize to the sequence of SEQ ID NO:38.

The nucleotide sequence encoding the Ala-Cpn10-EEE-cHis polypeptide ofthe invention may be as set forth in SEQ ID NO:40 or display sufficientsequence identity thereto to hybridize to the sequence of SEQ ID NO:40.

The nucleotide sequence encoding the Ala-Cpn10-cHis polypeptide of theinvention may be as set forth in SEQ ID NO:42 or display sufficientsequence identity thereto to hybridize to the sequence of SEQ ID NO:42.

The nucleotide sequence encoding the Cpn10-Δml polypeptide iscontemplated within the present invention and may be set forth in SEQ IDNO:4 or 5 or display sufficient sequence identity thereto to hybridizeto the sequence of SEQ ID NO:4 or 5.

The nucleotide sequence encoding the Cpn10-Δroof polypeptide iscontemplated in the present invention and may be set forth in SEQ IDNO:7 or 8 or display sufficient sequence identity thereto to hybridizeto the sequence of SEQ ID NO:7 or 8.

The nucleotide sequence encoding the Cpn10 β-barrel polypeptide iscontemplated in the present invention and may be set forth in SEQ IDNO:10 or display sufficient sequence identity thereto to hybridize tothe sequence of SEQ ID NO:10.

As for polypeptides discussed above, the term “variant” as used hereinrefers to substantially similar sequences. Generally, polynucleotidesequence variants encode polypeptides which possess qualitativebiological activity in common. Further, these polynucleotide sequencevariants may share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% sequence identity. Also included within themeaning of the term “variant” are homologues of polynucleotides of theinvention. A homologue is typically a polynucleotide from a differentspecies but sharing substantially the same activity.

Fragments of polynucleotides of the invention are also contemplated. Theterm “fragment” refers to a nucleic acid molecule that encodes aconstituent or is a constituent of a polynucleotide of the invention.Fragments of a polynucleotide, do not necessarily need to encodepolypeptides which retain biological activity. Rather the fragment may,for example, be useful as a hybridization probe or PCR primer. Thefragment may be derived from a polynucleotide of the invention oralternatively may be synthesized by some other means, for examplechemical synthesis. Polynucleotides of the invention and fragmentsthereof may also be used in the production of antisense molecules usingtechniques known to those skilled in the art.

Accordingly, the present invention contemplates oligonucleotides andfragments based on the sequences of the polynucleotides of the inventionfor use as primers and probes. Oligonucleotides are short stretches ofnucleotide residues suitable for use in nucleic acid amplificationreactions such as PCR, typically being at least about 10 nucleotides toabout 50 nucleotides in length, more typically about 15 to about 30nucleotides in length. Probes are nucleotide sequences of variablelength, for example between about 10 nucleotides and several thousandnucleotides, for use in detection of homologous sequences, typically byhybridization. The level of homology (sequence identity) betweensequences will largely be determined by the stringency of hybridizationconditions. In particular the nucleotide sequence used as a probe mayhybridize to a homologue or other variant of a polynucleotide disclosedherein under conditions of low stringency, medium stringency or highstringency. Low stringency hybridization conditions may correspond tohybridization performed at 50° C. in 2×SSC. There are numerousconditions and factors, well known to those skilled in the art, whichmay be employed to alter the stringency of hybridization. For instance,the length and nature (DNA, RNA, base composition) of the nucleic acidto be hybridized to a specified nucleic acid; concentration of salts andother components, such as the presence or absence of formamide, dextransulfate, polyethylene glycol etc; and altering the temperature of thehybridization and/or washing steps. For example, a hybridization filtermay be washed twice for 30 minutes in 2×SSC, 0.5% SDS and at least 55°C. (low stringency), at least 60° C. (medium stringency), at least 65°C. (medium/high stringency), at least 70° C. (high stringency) or atleast 75° C. (very high stringency).

In particular embodiments, polynucleotides of the invention may becloned into a vector. The vector may be a plasmid vector, a viralvector, or any other suitable vehicle adapted for the insertion offoreign sequences, their introduction into eukaryotic cells and theexpression of the introduced sequences. Typically the vector is aeukaryotic expression vector and may include expression control andprocessing sequences such as a promoter, an enhancer, ribosome bindingsites, polyadenylation signals and transcription termination sequences.

Antibodies

The present invention provides antibodies that selectively bind to theCpn10 polypeptides of the present invention, as well as fragments andanalogues thereof. Suitable antibodies include, but are not limited topolyclonal, monoclonal, chimeric, humanized, single chain, Fabfragments, and an Fab expression library. Antibodies of the presentinvention may act as agonists or antagonists of Cpn10 polypeptides, orfragments or analogues thereof.

Preferably antibodies are prepared from discrete regions or fragments ofthe Cpn10 polypeptides of the invention, in particular those involved inconferring immunomodulatory activity and/or partner or substratebinding. An antigenic Cpn10 polypeptide contains at least about 5, andpreferably at least about 10, amino acids.

Methods for the generation of suitable antibodies will be readilyappreciated by those skilled in the art. For example, an anti-Cpn10monoclonal antibody, typically containing Fab portions, may be preparedusing the hybridoma technology described in Antibodies-A LaboratoryManual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, N.Y.(1988).

In essence, in the preparation of monoclonal antibodies directed towardCpn10 polypeptides of the invention, fragments or analogues thereof, anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture may be used. These include thehybridoma technique originally developed by Kohler et al., Nature,256:495-497 (1975), as well as the trioma technique, the human B-cellhybridoma technique [Kozbor et al., Immunology Today, 4:72 (1983)], andthe EBV-hybridoma technique to produce human monoclonal antibodies [Coleet al., in Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R.Liss, Inc., (1985)]. Immortal, antibody-producing cell lines can becreated by techniques other than fusion, such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980);Hammerling et al., “Monoclonal Antibodies and T-cell Hybridomas” (1981);Kennett et al, “Monoclonal Antibodies” (1980).

In summary, a means of producing a hybridoma from which the monoclonalantibody is produced, a myeloma or other self-perpetuating cell line isfused with lymphocytes obtained from the spleen of a mammalhyperimmunized with a recognition factor-binding portion thereof, orrecognition factor, or an origin-specific DNA-binding portion thereof.Hybridomas producing a monoclonal antibody useful in practicing thisinvention are identified by their ability to immunoreact with thepresent recognition factor and their ability to inhibit specifiedtranscriptional activity in target cells.

A monoclonal antibody useful in practicing the present invention can beproduced by initiating a monoclonal hybridoma culture comprising anutrient medium containing a hybridoma that secretes antibody moleculesof the appropriate antigen specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well-known techniques.

Similarly, there are various procedures known in the art which may beused for the production of polyclonal antibodies to Cpn10 polypeptidesof the invention, or fragments or analogues thereof. For the productionof Cpn10 polyclonal antibody, various host animals can be immunized byinjection with a Cpn10 polypeptide, or a fragment or analogue thereof,including but not limited to rabbits, mice, rats, sheep, goats, etc.Further, the Cpn10 polypeptide or fragment or analogue thereof can beconjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA)or keyhole limpet hemocyanin (KLH). Also, various adjuvants may be usedto increase the immunological response, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Screening for the desired antibody can also be accomplished by a varietyof techniques known in the art. Assays for immunospecific binding ofantibodies may include, but are not limited to, radioimmunoassays,ELISAs (enzyme-linked immunosorbent assay), sandwich immunoassays,immunoradiometric assays, gel diffusion precipitation reactions,immunodiffusion assays, in situ immunoassays, Western blots,precipitation reactions, agglutination assays, complement fixationassays, immunofluorescence assays, protein A assays, andImmunoelectrophoresis assays, and the like (see, for example, Ausubel etal., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York). Antibody binding may be detected byvirtue of a detectable label on the primary anti-Cpn10 antibody.Alternatively, the anti-Cpn10 antibody may be detected by virtue of itsbinding with a secondary antibody or reagent which is appropriatelylabeled. A variety of methods are known in the art for detecting bindingin an immunoassay and are within the scope of the present invention.

Antibodies of the present invention can be used in diagnostic methodsand kits that are well known to those of ordinary skill in the art todetect qualitatively or quantify Cpn10 in a body fluid or tissue, oralternatively antibodies may be used in methods and compositions for thetreatment of various diseases, disorders and conditions.

The antibody (or fragment thereof) raised against a Cpn10 polypeptide ofthe invention or a fragment or analogue thereof has binding affinity forCpn10. Preferably, the antibody (or fragment thereof) has bindingaffinity or avidity greater than about 10⁵ M⁻¹, more preferably greaterthan about 10⁶ M⁻¹, more preferably still greater than about 10⁷ M⁻¹ andmost preferably greater than about 10⁸ M⁻¹.

In terms of obtaining a suitable amount of an antibody according to thepresent invention, one may manufacture the antibody(s) using batchfermentation with serum free medium. After fermentation the antibody maybe purified via a multistep procedure incorporating chromatography andviral inactivation/removal steps. For instance, the antibody may befirst separated by Protein A affinity chromatography and then treatedwith solvent/detergent to inactivate any lipid enveloped viruses.Further purification, typically by anion and cation exchangechromatography may be used to remove residual proteins,solvents/detergents and nucleic acids. The purified antibody may befurther purified and formulated into 0.9% saline using gel filtrationcolumns. The formulated bulk preparation may then be sterilized andviral filtered and dispensed.

Agonists and Antagonists

In addition to specific anti-Cpn10 antibodies, the polypeptides of thepresent invention, and fragments and variants thereof are particularlyuseful for the screening and identification of compounds and agents thatinteract with Cpn10. In particular, desirable compounds are those thatmodulate the immunomodulatory activity of Cpn10. Such compounds maymodulate by activating, increasing, inhibiting or preventing Cpn10immunomodulatory activity. Suitable compounds may exert their effect onCpn10 by virtue of either a direct (for example binding) or indirectinteraction.

Compounds which bind, or otherwise interact with Cpn10 polypeptides ofthe invention, and specifically compounds which modulate the activity ofCpn10, may be identified by a variety of suitable methods. Interactionand/or binding may be determined using standard competitive bindingassays or two-hybrid assay systems.

For example, the two-hybrid assay is a yeast-based genetic assay system(Fields and Song, 1989) typically used for detecting protein-proteininteractions. Briefly, this assay takes advantage of the multi-domainnature of transcriptional activators. For example, the DNA-bindingdomain of a known transcriptional activator may be fused to a Cpn10polypeptide of the invention, or fragment or variant thereof, and theactivation domain of the transcriptional activator fused to a candidateprotein. Interaction between the candidate protein and the Cpn10polypeptide, or fragment or variant thereof, will bring the DNA-bindingand activation domains of the transcriptional activator into closeproximity. Interaction can thus be detected by virtue of transcriptionof a specific reporter gene activated by the transcriptional activator.

Alternatively, affinity chromatography may be used to identify bindingpartners of Cpn10. For example, a Cpn10 polypeptide of the invention, orfragment or variant thereof, may be immobilized on a support (such assepharose) and cell lysates passed over the column. Proteins binding tothe immobilized Cpn10 polypeptide, fragment or variant can then beeluted from the column and identified. Initially such proteins may beidentified by N-terminal amino acid sequencing for example.

Alternatively, in a modification of the above technique, a fusionprotein may be generated by fusing a Cpn10 polypeptide, fragment orvariant to a detectable tag, such as alkaline phosphatase, and using amodified form of immunoprecipitation as described by Flanagan and Leder(1990).

Methods for detecting compounds that modulate Cpn10 activity may involvecombining a Cpn10 polypeptide with a candidate compound and a suitablelabeled substrate and monitoring the effect of the compound on Cpn10 bychanges in the substrate (may be determined as a function of time).Suitable labeled substrates include those labeled for colourimetric,radiometric, fluorimetric or fluorescent resonance energy transfer(FRET) based methods, for example.

Cpn10 polypeptides of the invention and appropriate fragments andvariants can be used in high-throughput screens to assay candidatecompounds for the ability to bind to, or otherwise interact with Cpn10.These candidate compounds can be further screened against functionalCpn10 to determine the effect of the compound on Cpn10 activity.

It will be appreciated that the above described methods are merelyexamples of the types of methods which may be employed to identifycompounds that are capable of interacting with, or modulating theactivity of, the Cpn10 polypeptides, and fragments and variants thereof,of the present invention. Other suitable methods will be known topersons skilled in the art and are within the scope of the presentinvention.

By the above methods, compounds can be identified which either activate(agonists) or inhibit (antagonists) Cpn10 activity. Such compounds maybe, for example, antibodies, low molecular weight peptides, nucleicacids or non-proteinaceous organic molecules.

Potential modulators of Cpn10 activity, for screening by the abovemethods, may be generated by a number of techniques known to thoseskilled in the art. For example, various forms of combinatorialchemistry may be used to generate putative non-peptide modulators.Additionally, techniques such as nuclear magnetic resonance (NMR) and Xray crystallography, may be used to model the structure of Cpn10polypeptides, fragments and variants and computer predictions used togenerate possible modulators.

Compositions and Routes of Administration

Cpn10 polypeptides and polynucleotides of the invention may be useful astherapeutic agents. These molecules find use, for example, in treatingor preventing a disease or condition in a subject, by administering atherapeutically effective amount of such a molecule to the subject.Typically such diseases and conditions are amenable to treatment bymodulation of the immune response in the subject. By way of example,such diseases and conditions may include acute or chronic inflammatorydiseases, asthma, allergy, multiple sclerosis, GVHD, and infectiousdiseases. The infectious disease may result from a bacterial or viralinfection. Accordingly, pharmaceutically useful compositions comprisingCpn10 polypeptides and polynucleotides for use in treating or preventingdiseases and conditions are contemplated.

Agonists and antagonists of Cpn10 polypeptides of the invention,including anti-Cpn10 antibodies, may also be useful as therapeuticagents. Accordingly, the present invention also contemplates methods oftreatment using such agonists and antagonists and pharmaceuticalcompositions comprising the same.

In general, suitable compositions for use in accordance with the methodsof the present invention may be prepared according to methods andprocedures that are known to those of ordinary skill in the art andaccordingly may include a pharmaceutically acceptable carrier, diluentand/or adjuvant.

Compositions may be administered by standard routes. In general, thecompositions may be administered by the parenteral (e.g., Intravenous,intraspinal, subcutaneous or intramuscular), oral or topical route.Administration may be systemic, regional or local. The particular routeof administration to be used in any given circumstance will depend on anumber of factors, including the nature of the condition to be treated,the severity and extent of the condition, the required dosage of theparticular compound to be delivered and the potential side-effects ofthe compound.

In general, suitable compositions may be prepared according to methodswhich are known to those of ordinary skill in the art and may include apharmaceutically acceptable diluent, adjuvant and/or excipient. Thediluents, adjuvants and excipients must be “acceptable” in terms ofbeing compatible with the other ingredients of the composition, and notdeleterious to the recipient thereof.

Examples of pharmaceutically acceptable carriers or diluents aredematerialized or distilled water; saline solution; vegetable based oilssuch as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil,sesame oils such as peanut oil, safflower oil, olive oil, cottonseedoil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils,including polysiloxanes, such as methyl polysiloxane, phenylpolysiloxane and methylphenyl polysolpoxane; volatile silicones; mineraloils such as liquid paraffin, soft paraffin or squalane; cellulosederivatives such as methyl cellulose, ethyl cellulose,carboxymethylcellulose, sodium carboxymethylcellulose orhydroxypropylmethylcellulose; lower alkanols, for example ethanol oriso-propanol; lower aralkanols; lower polyalkylene glycols or loweralkylene glycols, for example polyethylene glycol, polypropylene glycol,ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin;fatty acid esters such as isopropyl palmitate, isopropyl myristate orethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth orgum acacia, and petroleum jelly. Typically, the carrier or carriers willform from 10% to 99.9% by weight of the compositions.

The compositions of the invention may be in a form suitable foradministration by injection, in the form of a formulation suitable fororal ingestion (such as capsules, tablets, caplets, elixirs, forexample), in the form of an ointment, cream or lotion suitable fortopical administration, in a form suitable for delivery as an eye drop,in an aerosol form suitable for administration by inhalation, such as byintranasal inhalation or oral inhalation, in a form suitable forparenteral administration, that is, subcutaneous, intramuscular orintravenous injection.

For administration as an injectable solution or suspension, non-toxicparenterally acceptable diluents or carriers can include, Ringer'ssolution, isotonic saline, phosphate buffered saline, ethanol and 1,2propylene glycol.

Some examples of suitable carriers, diluents, excipients and adjuvantsfor oral use include peanut oil, liquid paraffin, sodiumcarboxymethylcellulose, methylcellulose, sodium alginate, gum acacia,gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine andlecithin. In addition these oral formulations may contain suitableflavoring and colorings agents. When used in capsule form the capsulesmay be coated with compounds such as glyceryl monostearate or glyceryldistearate which delay disintegration.

Adjuvants typically include emollients, emulsifiers, thickening agents,preservatives, bactericides and buffering agents.

Solid forms for oral administration may contain binders acceptable inhuman and veterinary pharmaceutical practice, sweeteners, disintegratingagents, diluents, flavorings, coating agents, preservatives, lubricantsand/or time delay agents. Suitable binders include gum acacia, gelatine,corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose orpolyethylene glycol. Suitable sweeteners include sucrose, lactose,glucose, aspartame or saccharine. Suitable disintegrating agents includecorn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthangum, bentonite, alginic acid or agar. Suitable diluents include lactose,sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate,calcium silicate or dicalcium phosphate. Suitable flavoring agentsinclude peppermint oil, oil of wintergreen, cherry, orange or raspberryflavoring. Suitable coating agents include polymers or copolymers ofacrylic acid and/or methacrylic acid and/or their esters, waxes, fattyalcohols, zein, shellac or gluten. Suitable preservatives include sodiumbenzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben,propyl paraben or sodium bisulphite. Suitable lubricants includemagnesium stearate, stearic acid, sodium oleate, sodium chloride ortalc. Suitable time delay agents include glyceryl monostearate orglyceryl distearate.

Liquid forms for oral administration may contain, in addition to theabove agents, a liquid carrier. Suitable liquid carriers include water,oils such as olive oil, peanut oil, sesame oil, sunflower oil, saffloweroil, arachis oil, coconut oil, liquid paraffin, ethylene glycol,propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol,glycerol, fatty alcohols, triglycerides or mixtures thereof.

Suspensions for oral administration may further comprise dispersingagents and/or suspending agents. Suitable suspending agents includesodium carboxymethylcellulose, methylcellulose,hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginateor acetyl alcohol. Suitable dispersing agents include lecithin,polyoxyethylene esters of fatty acids such as stearic acid,polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate,polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate andthe like.

The emulsions for oral administration may further comprise one or moreemulsifying agents. Suitable emulsifying agents include dispersingagents as exemplified above or natural gums such as guar gum, gum acaciaor gum tragacanth.

Methods for preparing parenterally administrable compositions areapparent to those skilled in the art, and are described in more detailin, for example, Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa., hereby incorporated by referenceherein.

The topical formulations of the present invention, comprise an activeingredient together with one or more acceptable carriers, and optionallyany other therapeutic ingredients. Formulations suitable for topicaladministration include liquid or semi-liquid preparations suitable forpenetration through the skin to the site of where treatment is required,such as liniments, lotions, creams, ointments or pastes, and dropssuitable for administration to the eye, ear or nose.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions. These may be prepared by dissolving theactive ingredient in an aqueous solution of a bactericidal and/orfungicidal agent and/or any other suitable preservative, and optionallyincluding a surface active agent. The resulting solution may then beclarified by filtration, transferred to a suitable container andsterilized. Sterilization may be achieved by: autoclaving or maintainingat 90° C.-100° C. for half an hour, or by filtration, followed bytransfer to a container by an aseptic technique. Examples ofbactericidal and fungicidal agents suitable for inclusion in the dropsare phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride(0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for thepreparation of an oily solution include glycerol, diluted alcohol andpropylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those described above in relation to thepreparation of drops. Lotions or liniments for application to the skinmay also include an agent to hasten drying and to cool the skin, such asan alcohol or acetone, and/or a moisturizer such as glycerol, or oilsuch as castor oil or arachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with a greasy or non-greasy basis. Thebasis may comprise hydrocarbons such as hard, soft or liquid paraffin,glycerol, beeswax, a metallic soap; a mucilage; an oil of natural originsuch as almond, corn, arachis, castor or olive oil; wool fat or itsderivatives, or a fatty acid such as stearic or oleic acid together withan alcohol such as propylene glycol or macrogols.

The composition may incorporate any suitable surfactant such as ananionic, cationic or non-Ionic surfactant such as sorbitan esters orpolyoxyethylene derivatives thereof. Suspending agents such as naturalgums, cellulose derivatives or inorganic materials such as silicaceoussilicas, and other ingredients such as lanolin, may also be included.

The compositions may also be administered in the form of liposomes.Liposomes are generally derived from phospholipids or other lipidsubstances, and are formed by mono- or multi-lamellar hydrated liquidcrystals that are dispersed in an aqueous medium. Any non-toxic,physiologically acceptable and metabolisable lipid capable of formingliposomes can be used. The compositions in liposome form may containstabilizers, preservatives, excipients and the like. The preferredlipids are the phospholipids and the phosphatidyl cholines (lecithins),both natural and synthetic. Methods to form liposomes are known in theart, and in relation to this specific reference is made to; Prescott,Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y.(1976), p. 33 et seq., the contents of which is incorporated herein byreference.

The compositions may be conjugated to an array of polyethylene glycol(PEG) derivatives. The addition of PEG to proteins (PEGylation) is awell established method for decreasing the plasma clearance rates ofproteins, thereby increasing their efficacy (Nucci et al., 1991, Adv.Drug Del. Rev. 6:133). Additional benefits of PEGylation may include,greater stability of proteins, decreased Immunogenicity, enhancedsolubility and decreased susceptibility to proteolysis (Sheffield W.2001, Curr Drug Targets Cardiovasc Haematol Disord. 1:1-22). PEGmolecules contain the basic repeating structure of —(OCH₃CH₂)n-OH andare classified into groups according to their molecular weight. PEGderivatives are conjugated to proteins to increase their hydrodynamicradius and in general, their increase in half-life is directly relatedto the size of the PEG chain attached (Sheffield W. 2001, Curr DrugTargets Cardiovasc Haematol Disord. 1:1-22).

The compositions may also be administered in the form of microparticles.Biodegradable microparticles formed from polylactide (PLA),polylactide-co-glycolide (PLGA), and epsilon-caprolactone ({acute over(ε)}-caprolactone) have been extensively used as drug carriers toincrease plasma half life and thereby prolong efficacy (R. Kumar, M.,2000, J Pharm Pharmaceut Sci. 3(2) 234-258). Microparticles have beenformulated for the delivery of a range of drug candidates includingvaccines, antibiotics, and DNA. Moreover, these formulations have beendeveloped for various delivery routes including parenteral subcutaneousinjection, intravenous injection and inhalation.

The compositions may incorporate a controlled release matrix that iscomposed of sucrose acetate isobutyrate (SAIB) and organic solvent ororganic solvents mixture. Polymer additives may be added to the vehicleas a release modifier to further increase the viscosity and slow downthe release rate. SAIB is a well known food additive. It is a veryhydrophobic, fully esterified sucrose derivative, at a nominal ratio ofsix isobutyrate to two acetate groups. As a mixed ester, SAIB does notcrystallize but exists as a clear viscous liquid. Mixing SAIB with apharmaceutically accepted organic solvent such as ethanol or benzylalcohol decreases the viscosity of the mixture sufficiently to allow forinjection. An active pharmaceutical ingredient may be added to the SAIBdelivery vehicle to form SAIB solution or suspension formulations. Whenthe formulation is injected subcutaneously, the solvent diffuses fromthe matrix allowing the SAIB-drug or SAIB-drug-polymer mixtures to setup as an in situ forming depot.

For the purposes of the present invention molecules and agents may beadministered to subjects as compositions either therapeutically orpreventively. In a therapeutic application, compositions areadministered to a patient already suffering from a disease, in an amountsufficient to cure or at least partially arrest the disease and itscomplications. The composition should provide a quantity of the moleculeor agent sufficient to effectively treat the patient.

The therapeutically effective dose level for any particular patient willdepend upon a variety of factors including: the disorder being treatedand the severity of the disorder; activity of the molecule or agentemployed; the composition employed; the age, body weight, generalhealth, sex and diet of the patient; the time of administration; theroute of administration; the rate of sequestration of the molecule oragent; the duration of the treatment; drugs used in combination orcoincidental with the treatment, together with other related factorswell known in medicine.

One skilled in the art would be able, by routine experimentation, todetermine an effective, non-toxic amount of agent or compound whichwould be required to treat applicable diseases and conditions.

Generally, an effective dosage is expected to be in the range of about0.0001 mg to about 1000 mg per kg body weight per 24 hours; typically,about 0.001 mg to about 750 mg per kg body weight per 24 hours; about0.01 mg to about 500 mg per kg body weight per 24 hours; about 0.1 mg toabout 500 mg per kg body weight per 24 hours; about 0.1 mg to about 250mg per kg body weight per 24 hours; about 1.0 mg to about 250 mg per kgbody weight per 24 hours. More typically, an effective dose range isexpected to be in the range about 1.0 mg to about 200 mg per kg bodyweight per 24 hours; about 1.0 mg to about 100 mg per kg body weight per24 hours; about 1.0 mg to about 50 mg per kg body weight per 24 hours;about 1.0 mg to about 25 mg per kg body weight per 24 hours; about 5.0mg to about 50 mg per kg body weight per 24 hours; about 5.0 mg to about20 mg per kg body weight per 24 hours; about 5.0 mg to about 15 mg perkg body weight per 24 hours.

Alternatively, an effective dosage may be up to about 500 mg/m².Generally, an effective dosage is expected to be in the range of about25 to about 500 mg/m², preferably about 25 to about 350 mg/m², morepreferably about 25 to about 300 mg/m², still more preferably about 25to about 250 mg/m², even more preferably about 50 to about 250 mg/m²,and still even more preferably about 75 to about 150 mg/m².

Typically, in therapeutic applications, the treatment would be for theduration of the disease state.

Further, it will be apparent to one of ordinary skill in the art thatthe optimal quantity and spacing of individual dosages will bedetermined by the nature and extent of the disease state being treated,the form, route and site of administration, and the nature of theparticular individual being treated. Also, such optimum conditions canbe determined by conventional techniques.

It will also be apparent to one of ordinary skill in the art that theoptimal course of treatment, such as, the number of doses of thecomposition given per day for a defined number of days, can beascertained by those skilled in the art using conventional course oftreatment determination tests.

Embodiments of the invention also contemplate the administration of apolynucleotide encoding Cpn10. In such situations the polynucleotide istypically operably linked to a promoter such that the appropriatepolypeptide sequence is produced following administration of thepolynucleotide to the subject. The polynucleotide may be administered tosubjects in a vector. The vector may be a plasmid vector, a viralvector, or any other suitable vehicle adapted for the insertion offoreign sequences, their introduction into eukaryotic cells and theexpression of the introduced sequences. Typically the vector is aeukaryotic expression vector and may include expression control andprocessing sequences such as a promoter, an enhancer, ribosome bindingsites, polyadenylation signals and transcription termination sequences.The nucleic acid construct to be administered may comprise naked DNA ormay be in the form of a composition, together with one or morepharmaceutically acceptable carriers.

Those skilled in the art will appreciate that in accordance with themethods of the present invention Cpn10 polypeptides of the invention maybe administered alone or in conjunction with one or more additionalagents. For example, a Cpn10 polypeptide of the invention may beadministered together with one or more agonists capable of stimulating aTLR receptor such as TLR4. Additionally, the present inventioncontemplates combination therapy using Cpn10 polypeptides of theinvention in conjunction with other therapeutic approaches to thetreatment of diseases and disorders. For example, Cpn10 polypeptides maybe useful in the treatment of viral diseases which are responsive totherapy with Type I interferons such as IFNβ or IFNα, and Cpn10polypeptides of the invention may be used in conjunction with IFNβ inthe treatment of autoimmune diseases such as multiple sclerosis.

For such combination therapies, each component of the combinationtherapy may be administered at the same time, or sequentially in anyorder, or at different times, so as to provide the desired effect.Alternatively, the components may be formulated together in a singledosage unit as a combination product. When administered separately, itmay be preferred for the components to be administered by the same routeof administration, although it is not necessary for this to be so.

The present invention will now be described with reference to specificexamples, which should not be construed as in any way limiting the scopeof the invention.

EXAMPLES Example 1 Genetic Parameters Used for the Production of Cpn10Polypeptides

Table 2 describes the genetic parameters, specifically the expressionsystems, (i.e. plasmid names, antibiotic selection and host cells) usedfor the production of the Cpn10 polypeptides listed below,

TABLE 2 Description of genetic parameters for the production of theCpn10 polypeptides Cpn10 polypeptide Plasmid Name Production SystemAla-Cpn10 Ala-Cpn10_pPL550 XL1-Blue cells with pPL550 AmpR X-Cpn10X-Cpn10_pPL550 XL1-Blue cells with pPL550 AmpR Ala-Cpn10-ΔmlAla-Cpn10-Δml_pPL550 XL1-Blue cells with pPL550 AmpR Ala-Cpn10-Ala-Cpn10- XL1-Blue cells with pPL550 AmpR Δroof Δroof_pPL550Ala-Cpn10-β- Ala-Cpn10-β- BL21(DE3)STAR cells with pET23a barrelbarrel_pET23a AmpR Gly-Cpn10 Gly-Cpn10 _pET30a BL21(DE3)STAR cells withpET30a KanR GroES GroES_pET11a BL21(DE3)STAR cells with pET11a AmpRAla-Cpn10-IFI Ala-Cpn10-IFI_pPL550 XL1-Blue cells with pPL550 AmpRAla-Cpn10-III Ala-Cpn10-III_pPL550 XL1-Blue cells with pPL550 AmpRAla-Cpn10- Ala-Cpn10-EEE-cHis_pET23a BL21(DE3)STAR cells with pET23aEEE-cHis AmpR Ala-Cpn10-cHis Ala-Cpn10-cHis_pET23a BL21(DE3)STAR cellswith pET23a AmpR Cpn10-NtermES Cpn10-NtermES_pET23a BL21(DE3)STAR cellswith pET23a AmpR

Example 2 Process for Producing Cpn10 Polypeptides

To further define the production process of Cpn10, Ala-Cpn10 isexemplified below with regard to the production process.

Firstly, a heat-inducible expression plasmid encoding human Cpn10 withan additional N-terminal alanine residue (Ala-Cpn10_pPL550) was obtainedfrom Somodevilla-Torres et al. (2003, Prot. Exp. Purif. 32:276-287).Then, the plasmid vector was transformed into the E. coli strainXL1-Blue (Stratagene), and a master cell bank was established from asingle selected clone.

Ala-Cpn10 was then produced in E. coli essentially as described by Ryanet al. (1995, J Biol Chem 270:22037-22043). In addition, the materialthat did not bind Macro-Prep High Q (BioRad) was further purified byS-Sepharose and then Gel-Filtration (Superdex 200, AmershamBiosciences). Purified Cpn10 in a 50 mM Tris-HCl (pH 7.6) and 150 mMNaCl buffer, was filtered through an Acrodisc with a 0.2 mm Mustang Emembrane according to the manufacturer's instructions (Pall Corporation,Ann Arbor, Mich. Cat No. MSTG5E3) to remove residual endotoxins and wasstored at −70° C. The purity of Cpn10 was determined to be >99% bySDS-PAGE. Aliquots were thawed once prior to use.

Most of human Cpn10 polypeptides showed the same molar activity as E.coli GroES in GroEL-mediated rhodanese refolding assays (Brinker et al.,2001, Cell, 107 223-233) (data not shown). LPS contamination of Cpn10was determined by the Limulus Amebocyte Lysate assay (BioWhittaker,Walkersvllle, Md.) to be <0.03 EU/mg of purified Cpn10 protein.

The authenticity of Cpn10 polypeptides obtained from the productionprocess as described above was assessed on a batch by batch basis bymass spectrometry. As shown in table 3 below the predicted andcalculated masses are in agreement.

TABLE 3 Mass Spectrometry Data and Theoretical pl for Cpn10polypeptides. Predicted Calculated Cpn10 polypeptides mass (Da) mass(Da) Ala-Cpn10 10871.5 10871.6 10871.5 10873.0 X-Cpn10 10800.5 10799.3Ala-Cpn10-Δml 9200.6 9200.0 Ala-Cpn10-Δroof 10201.8 10202.0Ala-Cpn10-β-barrel 8530.8 8531.0 Gly-Cpn10 10857.5 10857.0 GroES 10386.910386.0 Ala-Cpn10-IFI 10887.5 10885.8 Ala-Cpn10-III 10853.5 10851.9Ala-Cpn10-EEE-cHis 11966.5 11966.0 Ala-Cpn10-cHis 11936.7 11936.0Cpn10-NtermES 10295.9 10295.0

Example 3 RAW264-HIV-LTR-LUC Bioassay to Determine ImmunomodulatoryActivity Cpn10 Polypeptides

Immunomodulatory activity of Cpn10 polypeptides were tested using theRAW264-HIV-LTR luciferase bioassay essentially as described inInternational Patent Application No. PCT/AU2005/000041. This assay, inthe presence of lipopolysaccharide (LPS), measures the ability of Cpn10or the variant, mutant or derivative thereof to modulate signalling fromthe Toll-like receptor TLR4.

RAW264-HIV-LTR-LUC cells were cultured in the presence of G418 (200mg/ml) for 5 days after recovery from liquid nitrogen and grown assuspension cultures in 75 cm2 flasks (Greiner Labortechnik,Frickenhausen, Germany). RAW264-HIV-LTR-LUC cells were disaggregated byrepeated pipetting and plated at 2.5×105 cells/well in 24-well platesand incubated overnight (37° C. and 5% CO2). Crude LPS from E. coli(Cat. No. L-6529, Strain 055:B5, Sigma) and ultra-pure LPS from E. coli(Cat. No. tlrl-pelps, Strain 0111:B4, Invivogen) were dissolved insterile distilled water and stored at 4° C. in glass vials at 1 mg/ml or5 mg/ml respectively. Immediately prior to use, the solution wasvigorously vortexed before aliquots were taken. Cpn10 was pre-incubatedwith cells for 2 h prior to the addition of LPS at the indicatedconcentrations. Following a further 2 h incubation, the adherent cellswere processed for the luciferase assay (Luciferase Assay System,Promega, Madison, Wis.). Luciferase activity was measured using either aTurner Designs Luminometer TD 20/20 (RLU) or a Perkin-Elmer WallaceVictor 2 Multilabel Counter (CPS).

Example 4 Analysis of Co-chaperone Activity for Cpn10 Polypeptides Usingan in vitro GroEL-mediated Rhodanese Refolding Assay

The ability of Cpn10 polypeptides to act as molecular chaperones andfold proteins in conjunction with GroEL was determined by assaying forrhodanese refolding in vitro utilizing a method adapted from Weber F,and Hayer-Hartl M. K. (Chaperonin Protocols, Ed Schneider C, HumanaPress Inc., 2000, p117-126). Native bovine rhodanese (30 μM, SIGMA) wasdenatured in 20 mM MOPS-KOH (pH7.5), 100 mM KCl and 20 mM MgCl₂ (bufferA) containing 5M Guanidine HCl and 8 mM DTT then subsequently diluted(75-fold) from denaturant into buffer A containing GroEL (400 nM), suchthat the final concentration of rhodanese was 400 nM. GroEL rapidly andstably binds denatured rhodanese (D-Rho) whereas in buffer alone, D-Rhomis-folds and aggregates (ie inefficient spontaneous refolding). Theaddition of Cpn10 (see below) and ATP (20.6 mM) to preformed, stablecomplexes of GroEL-bound rhodanese permits efficient refolding toproceed. In the absence of Cpn10, the addition of ATP causes D-Rho tocycle on and off GroEL in a folding incompetent manner leadingeventually to misfolding and aggregation (this reaction serves as asuitable assay blank). Each folding reaction has a total volume of 290μl, at specific time points (ie 0, 15, 30, 45, 60, 75, 90 mins) 30 μLaliquots are removed and combined with 70 μL of rhodanese activity assaymixture (57.1 mM KH₂PO₄ (PH7.5), 71.4 mM EDTA, 71.4 mM Na thiosulfateand 71.4 mM KCN) for 6 min. Prior to the initiation of refoldingreactions with ATP, a 30 μl aliquot is taken as a T=0 min of refoldingtime point. EDTA within the rhodanese activity assay mixture chelatesMg²⁺ ions, which prevents GroEL binding ATP, the result is an immediatestopping of the folding reaction. Subsequently, rhodanese activity isstopped after 6 min by the addition of 50 μL of 15% (v/v) formaldehyde(final concentration 5% v/v).

Rhodanese catalyses the formation of thiocyanide (‘Rhodanid’) fromthiosulfate and cyanide. Thiocyanide is easily detected colorimetrically(Absorbance 450 nm) by the formation of its red iron complex in thepresence of Ferric Nitrate. Rhodanese activity measurements (150 μl) aredeveloped by the addition of 150 μl of Ferric Nitrate reagent (164.5 mMferric nitrate and 9.2% v/v nitric acid). Rhodanese activitymeasurements are read at A450 nm in 96 well microplates.

A typical rhodanese folding reactions follow an exponential incline inrhodanese activity (ie folded rhodanese) with time to a maximum yield offolded rhodanese. At constant amounts of GroEL (400 nM) and rhodanese(400 nM), a linear relationship is observed (between rhodanese activityand time) with increasing amounts of Cpn10 until an equal molarconcentration of Cpn10 (7 mer) to GroEL (14 mer) is reached (ie 400 nM).At concentrations of Cpn10 above 400 nM, the increase in rhodaneseactivity rapidly reaches a maximum. The assay consists of five standards(in duplicate) and test samples (in duplicate). The concentrations ofCpn10 standards are 0 nM, 140 nM, 250 nM, 280 nM and 350 nM. Rhodaneseactivity (ie Cpn10 activity) measurements from the 30, 45, 60, 75 and 90min time points are averaged. The 0 nM Cpn10 standard serves as asuitable measurement of the assays' background activity; therefore theabsorbance value for the 0 nM Cpn10 standard is subtracted from allother calculated absorbance values (or activity values). Followingbackground correction, the absorbance value for the 280 nM Cpn10standard is nominated as 100% activity and all other absorbance valuesare converted to a relative % activity based on the 100% standard.Outlier data points are removed by comparison of duplicatemeasurements, >30% deviation between duplicates is consideredunacceptable. Utilizing the acceptable data, a linear calibration curveis generated with five standard concentrations 0 nM Cpn10 (0% Activity),140 nM Cpn10 (50% Activity), 250 nM Cpn10 (89.3% Activity), 280 nM Cpn10(100% Activity) and 350 nM Cpn10 (125% Activity). Rhodanese activity (ieCpn10 activity) is plotted against Cpn10 concentration. For correctionof assay bias, the % activity values from the test samples arerecalculated using the equation generated from the linear calibrationcurve.

Concentrations of chaperonins are calculated using the oligomericmolecular weights (MW) of the proteins while rhodanese is calculatedusing the monomeric MW; i.e E. coli GroEL 14 mer (SwissProtP06139)=800,766.4 g/mol, Human Cpn10 7 mer (SwissProt Q04984)=76,100.5g/mol and Bovine rhodanese 1 mer (SwisProt P00586)=33,164.6 g/mol.

Table 4 shows the refolding activity of the Cpn10 polypeptides on abatch by batch basis and calculated as a percentage of Ala-Cpn10activity,

TABLE 4 Refolding Activity of Cpn10 polypeptides Refolding activityCpn10 Polypeptides (% of Ala-Cpn10) Ala-Cpn10 100 100 X-Cpn10 105Ala-Cpn10-Δml 0 Ala-Cpn10-Δroof 68 Ala-Cpn10-β-barrel 0 Gly-Cpn10 107GroES 109 Ala-Cpn10-IFI 114 Ala-Cpn10-III 41 Ala-Cpn10-EEE-cHis 0Ala-Cpn10-cHis 40 Cpn10-NtermES 12

Example 5 E. coli GroES does not Inhibit LPS-mediated HIV-LTR Activation

Recombinant E. coli GroES was purified and shown to be essentially freeof endotoxin contamination 0.14 EU/mg (see FIG. 3K). Purified GroES wastested in the RAW264.7-HIV-LTR-LUC inhibition assay side by side withAla-Cpn10 as described above. As shown in FIG. 2, GroES did not inhibitLPS-induced activation of HIV-LTR at any of the tested concentrations(25-100 μg/ml). These results confirm that the immunomodulatory activityobserved for Cpn10 is a real and significant biological effect.

Example 6 Construction of Human Cpn10 Mutants

Site-specific Mutants of IML₂₃₋₂₅

The hydrophobic IML moiety (residues 23-25) of the mobile loop regionwas mutated to alter the strength of interaction between Cpn10 and Cpn60(see Table 1). IML was replaced with the charged tripeptide EEE which ispredicted to perturb interaction with Cpn60. IML was also mutated toeither III or IFI moieties both which are predicted to increasehydrophobicity and thereby potentially, strengthen the interaction ofCpn10 with Cpn60. An SDS-PAGE gel showing purification ofAla-Cpn10-EEE-cHis, Ala-Cpn10-IFI, Ala-Cpn10-III and is presented inFIGS. 3C, E and F.

Ala-Cpn10-III, Ala-Cpn10-IFI and Ala-Cpn10-EEE-cHis were generated byQuick Change Site-Directed Mutagenesis (Stratagene) according to themanufacturer's instructions utilizing the complimentary pairs of primersas set forth in table 1. For Ala-Cpn10-III and Ala-Cpn10-IFI, theAla-Cpn10_pPL550 plasmid was used as the DNA template. ForAla-Cpn10-EEE-cHis, the Ala-Cpn10-cHis_pET23 plasmid was used as the DNAtemplate (see below),

Ala-Cpn10

The amino acid sequence predicted to comprise Ala-Cpn10 is set forth inSEQ ID NO:21. A synthetic DNA sequence encoding Ala-Cpn10 (SEQ ID NO:22)was inserted into the pPL550 plasmid at the NcoI and EcoRI sites(Somodevilla-Torres et al., 2003, Prot. Exp. Purif. 32:276-287). AnSDS-PAGE gel showing purification of Ala-Cpn10 in batch CH001 and CH003is presented in FIGS. 3A and B respectively.

Ala-Cpn10-cHis

The amino acid sequence predicted to comprise Ala-Cpn10-cHis is setforth in SEQ ID NO:41. A synthetic DNA sequence encoding Ala-Cpn10-cHis(SEQ ID NO:42) was prepared by insertion of the Ala-Cpn10 DNA sequence(SEQ ID NO:22) minus the stop codon into the pET23a plasmid (Novagen) atthe Ndel and Xhol sites. The cloning enables a C-terminal hexahistidine(SEQ ID NO:45) tag to be present on Ala-Cpn10. An SDS-PAGE gel showingpurification of Ala-Cpn10-cHis is presented in FIG. 3D.

Ala-Cpn10-Δml

16 amino acids were deleted from the mobile loop region (SEQ ID NO:12)of Cpn10 to generate the 86 amino acid variant designated Ala-Cpn10-Δml(SEQ ID NO: 24). A synthetic DNA sequence encoding Ala-Cpn10-Δml (SEQ IDNO: 25) was inserted into the pPL550 plasmid at the NcoI and EcoRI sites(Somodevilla-Torres et al., 2003, Prot. Exp. Purif. 32:276-287). As themobile loop is situated in the middle of the Cpn10 polypeptide chain,two residues (one at either end) of the mobile loop were retained tojoin the N-terminal fragment with the C-terminal fragment and ensureproper folding and assembly of the heptamer.

An SDS-PAGE gel showing purification of Ala-Cpn10-Δml is presented inFIG. 3G. Partial glutaraldehyde cross-linking of Ala-Cpn10-Δml (FIG. 3H,lane 2) shows 7 distinct bands on silver stained 4-12% SDS-PAGE gel,confirming the heptameric structure of the molecule. An amount of 580 μgof Ala-Cpn10-Δml in PBS (pH 7.4) was incubated with 0.01% (w/w)glutaraldehyde (APS) in a total volume of 300 μl at 25° C. for 30 min.Reactions were quenched by the addition of 15 μl of 2M Tris-HCl (pH8.0). An aliquot of 100 μl of the reaction mixture was resolved on aSuperdex 200 HR10/30 (GE Biosciences) size exclusion column in phosphatebuffered saline (PBS) at a flow rate of 0.5 ml min⁻¹. The peak elutingat the same retention time as the non-cross-linked Cpn10 oligomer wascollected in two 0.5 ml fractions and subsequently analyzed by SDS-PAGEand silver staining.

Ala-Cpn10-Δroof

7 amino acids were deleted from the β-hairpin region (SEQ ID NO:13) togenerate the 95 amino acid variant designated Ala-Cpn10-Δroof. Asynthetic DNA sequence encoding Ala-Cpn10-Δroof (SEQ ID NO:27). wasinserted into the pPL550 plasmid at the NcoI and EcoRI sites(Somodevilla-Torres et al., 2003, Prot. Exp. Purif. 32; 276-287). Theamino acid sequence of Ala-Cpn10-Δroof is set forth in SEQ ID NO:26.Further, the polynucleotide encoding this polypeptide is set forth inSEQ ID NO:27. An SDS-PAGE gel showing purification of Ala-Cpn10-Δroof ispresented in FIG. 3I.

Ala-Cpn10-β-barrel

The amino acid sequence predicted to comprise Ala-Cpn10-β-barrel is setforth in SEQ ID NO:28. An SDS-PAGE gel showing purification ofCpn10-β-barrel is presented in FIG. 3D. A synthetic DNA sequenceencoding Ala-Cpn10-β-barrel (SEQ ID NO:29) was inserted into the pPL550plasmid at the NcoI and EcoRI sites (Somodevilla-Torres et al., 2003,Prot. Exp. Purif. 32:276-287). An SDS-PAGE gel showing purification ofAla-Cpn10-β-barrel is presented in FIG. 3J.

Gly-Cpn10

The amino acid sequence predicted to comprise Gly-Cpn10 is set forth inSEQ ID NO:30. A synthetic DNA sequence encoding Gly-Cpn10 (SEQ ID NO:31)was inserted into the pET30a plasmid. An SDS-PAGE gel showingpurification of Gly-Cpn10 is presented in FIG. 3N.

X-Cpn10

The amino acid sequence predicted to comprise X-Cpn10 is set forth inSEQ ID NO:23. A synthetic DNA sequence encoding X-Cpn10 (SEQ ID NO:44)was inserted into the pPL550 plasmid at the NcoI and EcoRI sites(Somodevilla-Torres et al., 2003, Prot. Exp. Purif. 32:276-287). AnSDS-PAGE gel showing purification of X-Cpn10 is presented in FIG. 3M.

GroES

The amino acid sequence predicted to comprise E. coli GroES (SwissProtP05380) is set forth in SEQ ID NO:11. A synthetic DNA sequence encodingGroES (SEQ ID NO:34) was inserted into the pET11a plasmid. An SDS-PAGEgel showing purification of GroES is presented in FIG. 3K.

Cpn10-NtermES

The amino acid sequence predicted to comprise Cpn10-NtermES is set forthin SEQ ID NO:14. The Cpn10-NtermES protein was made by replacingresidues 1-AGQAFRKFL-9 (SEQ ID NO:33) of human X-Cpn10 (SEQ ID NO:23)with residues 1-MNIR-4 (SEQ ID NO:46) of E. coli GroES (SEQ ID NO:11). Asynthetic DNA sequence encoding Cpn10-NtermES (SEQ ID NO:43) wasinserted into the pET23a plasmid. An SDS-PAGE gel showing purificationof Cpn10-NtermES is presented in FIG. 3L.

Example 7 Activity of IML Tripeptide Mutants of Cpn10

Mutants of the IML tripeptide of the mobile loop (crucial to interactionwith Cpn60 and therefore to protein folding) were generated to eitherperturb or strengthen the interaction of the mobile loop with Cpn60 (seeExample 6).

The EEE mobile loop Ala-Cpn10 mutant protein (Ala-Cpn10-EEE-cHis)abolished the ability of Cpn10 to function with GroEL (E. coli Cpn60)during the process of rhodanese refolding in vitro, while the III andIFI mutants remained active (see Table 3).

The results indicate that the affinity of Ala-Cpn10-EEE-cHis for GroELis indeed significantly reduced. In contrast to the protein foldingassay, the RAW264.7-HIV-LTR-LUC inhibition assay demonstrated that alltripeptide mutants (including Ala-Cpn10-cHis) are able to modulate TLR4signalling, with activity similar to the Ala-Cpn10, indicating that themobile loop (and therefore Cpn60) is not important for thisimmunomodulatory activity of Cpn10 (see FIGS. 4B, D, F and H).

Example 8 Ala-Cpn10-Δml does not Cooperate with GroEL in RhodaneseRefolding

To confirm that the mobile loop region (and therefore Cpn60) is notrequired for immunomodulatory activity, 16 amino acids were deleted fromthe mobile loop region (SEQ ID NO:12) to generate the 86 amino acidvariant designated Ala-Cpn10-Δml (see Example 6). The amino acidsequence of Ala-Cpn10-Δml is set forth in SEQ ID NO:24.

Ala-Cpn10-Δml was tested for its ability to function productively withGroEL (E. coli Cpn60) in the process of rhodanese refolding. As apositive control, Ala-Cpn10 was included in the same assay and resultedin an activity of ˜100% (see table 3), The activity of Ala-Cpn10-Δml wasmeasured as ˜0% activity, indicating that it does not interact withGroEL and therefore cannot function as a co-chaperone during the processof protein folding. Both the Ala-Cpn10 and Ala-Cpn10-Δml polypeptideswere tested at equal molar concentration.

Example 9 Ala-Cpn10-Δml Inhibits LPS-Induced Activation of HIV LTR

Ala-Cpn10-Δml was tested using the RAW264.7-HIV-LTR-LUC inhibition assayside by side with Ala-Cpn10. In this assay a luciferase reporter islinked indirectly to NFκB signal transduction. NFκB is the primarytranscription factor induced by LPS. Luciferase activity is measured asrelative light units (RLU) or counts per second (CPS) depending on theinstrumentation used. As shown in FIGS. 5A and 5B, Ala-Cpn10-Δmlinhibited LPS-induced activation of HIV-LTR between the concentrationsof 1 and 100 μg/ml. Although this is a single assay, two replicateexperiments were set up on separate microtitre plates and demonstratedthe same activity. In this data set, Ala-Cpn10-Δml appeared to possessconsistently greater inhibitory activity as compared with Ala-Cpn10.

As described above in Example 8, Ala-Cpn10-Δml was not able to functionas a co-chaperone for GroEL in the process of rhodanese refolding.However, when used in the RAW264.7-HIV-LTR-LUC inhibition assay in thepresence of LPS, Ala-Cpn10 levels of activity of Ala-Cpn10-Δml wasobserved. That is, Ala-Cpn10-Δml dose-dependently inhibited LPS-inducedactivation of the HIV-LTR reporter. These results clearly rule out aninvolvement of Cpn60 in the ability of Cpn10 to modulate TLR4signalling.

Example 10 Ala-Cpn10-Δroof Inhibits LPS-induced Activation of HIV LTR

The β-hairpin roof loop region (see FIG. 1) of Cpn10 contains a netpositive charge in mammals but predominantly a net negative charge inbacteria (for example as represented by E. coli GroES). Intriguingly,bacteriophage T4 also contains a specialized Cpn10 (Gp31) that functionstogether with E. coli GroEL to fold the T4 major capsid protein Gp23,Neither GroES nor Cpn10 can fulfill this function. A major differencebetween Gp31 and Cpn10/GroES is that Gp31 completely lacks the roofβ-hairpin loop, possibly accounting for the unusual function andabilities of Gp31 (Hunt et al., 1997, Cell 90:361-371).

To determine the contribution of the roof β-hairpin region to Cpn10immunomodulatory activity, 7 amino acids were deleted from the β-hairpinregion (SEQ ID NO:13) to generate the 95 amino acid variant designatedAla-Cpn10-Δroof (see Example 6). The amino acid sequence ofAla-Cpn10-Δroof is set forth in SEQ ID NO:26.

Ala-Cpn10-Δroof was tested in the RAW264.7-HIV-LTR-LUC inhibition assayin the presence of LPS, side by side with Ala-Cpn10 and E. coli GroES.As shown in FIG. 6, Ala-Cpn10-Δroof inhibited LPS-induced activation ofHIV-TLR between the concentrations of 50 and 100 μg/ml. Although this isa single assay, two replicate experiments were set up on separatemicrotitre plates and demonstrated the same activity. In this data set,Ala-Cpn10-Δroof appeared to possess consistently ˜80% of the activity ofAla-Cpn10. These results demonstrate that modulation of TLR4 signallingby Cpn10 can occur in the absence of a functional roof β-hairpin regionof the molecule.

Example 11 Ala-Cpn10-β-barrel Mutant Exhibits Immunomodulatory Activity

As a confirmation of the above data described in Examples 9 and 10, theinventors generated a human Ala-Cpn10 mutant which lacks both the mobileloops and the β-hairpin roof loops (termed “Ala-Cpn10-β-barrel”; SEQ IDNO:28; see Example 6). Interestingly, the Ala-Cpn10-β-barrel mutant runsas a slightly larger entity on gel filtration chromatography comparedwith Ala-Cpn10 and the Ala-Cpn10-Δml mutant. This may imply that theroof helps to hold the subunits in a tightly bound conformation. Incomparison, the mobile loop destabilizes the heptamer leading to moreefficient disassembly; despite this the heptamer is energetically morefavored than the disassembled monomers.

The Ala-Cpn10-β-barrel polypeptide was tested in theRAW264.7-HIV-LTR-LUC inhibition assay in the presence of LPS, side byside with Ala-Cpn10 and E. coli GroES. As shown in FIG. 7, this mutantdisplayed approximately 50% of the Ala-Cpn10 activity in modulating TLR4signalling, suggesting that immunomodulatory activity may be partlycontributed to the β-hairpin roof loops or might be attributed tostability of the heptamer. The results shown are reflective of twoindependent experiments.

Example 12 Immunomodulatory Activity of an N-terminal Cpn10 Mutant

The Cpn10 N-terminus is known to assist in targeting to themitochondrial matrix (following synthesis in the cytosol). However,while most mitochondrial matrix proteins bear a cleavable N-terminaltargeting sequence, the Cpn10 N-terminus is not cleaved indicating thatit may have a further function.

The inventors have investigated the ability of an N-terminal mutant ofhuman Cpn10 to modulate immune reactivity in vitro. The mutant tested(referred to herein as “Cpn10-NtermES”) bears the N-terminal sequence“MNIR” (SEQ ID NO:46) from E. coli GroES in place of the humanN-terminal sequence “MAGQAFRKFL” (SEQ ID NO:32). The amino acid sequenceof Cpn10-NtermES is provided in SEQ ID NO:14.

The Cpn10-NtermES, showed only ˜12% of Ala-Cpn10 activity in chaperonemediated rhodanese refolding (with GroEL; see Table 3). It was howeverconfirmed by gel filtration chromatography that Cpn10-NtermES is aheptamer with intact mobile loops for GroEL (Cpn60) binding (data notshown).

As shown in FIGS. 8A and 8B, the Cpn10-NtermES mutant lost the abilityto immunomodulate LPS-induced activation of HIV-TLR in contrast to theactivity observed with Ala-Cpn10. This indicates that the N-terminus ofCpn10 is required for Cpn10 immunomodulatory activity involving TLR4.Therefore, ES-Nterm-Cpn10, like E. coli (GroES), was unable to suppressNF-κB induced by LPS (ie TLR4 modulation) (see FIG. 8B).

Example 13 Reduced Ability of X-Cpn10 to Inhibit LPS-induced Activationof HIV LTR

X-Cpn10 was tested using the RAW264.7-HIV-LTR-LUC inhibition assay sideby side with Ala-Cpn10. As discussed in example 6, X-Cpn10 lacks theextra N terminal alanine residue and an acetyl group which is present innative human Cpn10. In this assay a luciferase reporter is linkedindirectly to NFκB signal transduction. NFκB is the primarytranscription factor induced by LPS. Luciferase activity is measured asrelative light units (RLU) or counts per second (CPS) depending on theinstrumentation used. As shown in FIGS. 9A and 9B, X-Cpn10 partiallyinhibited LPS-induced activation of HIV-LTR (approximately 50% of Ala-or Gly-Cpn10 activity). In this data set, it appears that additionalresidues on the N terminus of Cpn10 such as an alanine may contribute tothe immunomodulatory activity of TLR4 signalling.

Example 14 Gly-Cpn10 Inhibits LPS-induced Activation of HIV LTR

Gly-Cpn10 was tested using the RAW264.7-HIV-LTR-LUC inhibition assayside by side with Ala-Cpn10. As discussed in example 6, Gly-Cpn10contains a glycine residue which replaced the extra N terminal alanineresidue of Ala-Cpn10. In this assay a luciferase reporter is linkedindirectly to NFκB signal transduction. NFκB is the primarytranscription factor induced by LPS. Luciferase activity is measured asrelative light units (RLU) or counts per second (CPS) depending on theinstrumentation used. As shown in FIGS. 10A and 10B, Gly-Cpn10 inhibitedLPS-induced activation of HIV-LTR to a greater extend than Ala-Cpn10.

As shown in the FIG. 16, an acetyl group is more structurally similar toa glycine residue than an alanine residue. It is therefore contemplatedthat the activity of the Acetyl-Cpn10 polypeptide (i.e. native Cpn10) issimilar to Gly-Cpn10.

Example 15 Use of Ultra Pure LPS in RAW264.7-HIV-LTR-LUC InhibitionAssay

The data as shown in FIGS. 5-7 and 10 were generated by using crude LPSin the abovementioned RAW264.7-HIV-LTR-LUC inhibition assay. In FIGS. 11to 14, ultra pure LPS was used which is specific for TLR4. The resultsfrom FIGS. 11 (Ala-Cpn10Δml), 12 (Ala-Cpn10Δroof), 13(Ala-Cpn10-β-barrel) and 14 (Gly-Cpn10) are very similar to theircounterparts as represented in FIGS. 5-7 and 10. This shows that theassays that are used and disclosed herein for the generation of theimmunomodulatory activity of Cpn10 are specific for TLR4.

Example 16 Mouse Endotoxemia Study

A mouse endotoxemia study was undertaken to determine whether the invitro immunomodulatory activity of various Cpn10 polypeptides (i.e.Ala-Cpn10-Δml, Ala-Cpn10-Δroof, and X-Cpn10) reflects the in vivoactivity using a mouse model of sepsis.

By systematically changing or deleting hypothesized active regions ofthe Cpn10 molecule, and by testing homologues of Cpn10 from the numerousbiological kingdoms, the minimal structural regions and/orsequence-based motifs necessary for optimal activity can be described,leading ultimately to the ability to design a more potent molecule fortherapeutic use. To date several variations or mutations in Cpn10 havebeen made in order to examine the importance of these regions forimmunomodulatory activity (see FIG. 1).

From these in vitro studies, it has been observed that relative toAla-Cpn10, constructs with a deletion in the mobile loop or roof loopregion of Cpn10 (i.e. Ala-Cpn10-Δml and Ala-Cpn10-Δroof respectively),showed comparable activity in terms of reducing NFκB activation inresponse to ligation of TLR4 with LPS. On the other hand, thenon-acetylated Cpn10 (X-Cpn10) showed significantly reduced ability todown-modulate NFκB activity using this in vitro assessment of activity.

In the present study a number of Cpn10 variants were found to havesimilar activity to Ala-Cpn10 in an in vivo model of inflammation (i.e.endotoxemia). The mouse endotoxemia model measures the ability of Cpn10to reduce LPS-induced inflammatory cytokine production.

Example 16a Material and Methods for the Mouse Endotoxemia Study

The following materials and methods used in the mouse endotoxemia studyare described below in examples 17a(1) to 17(a)2.

Example 16a(1) Mice Used for the Endotoxemia Study

The study was conducted on 84 female Balb/c mice. All were adult (>9weeks of age, average weight ˜20 g (0.02 kg)), and divided into twelvegroups with seven mice per group (see Table 5). The mice were housedwith a 12/12 light/dark cycle and have access to standard laboratorychow (Specialty Feeds, Glen Forrest, Australia) and water ad lib. Theweight of each mouse was measured prior to the start of injections. Thegroups received the following injections via intravenous (IV) route intothe tail vein as shown below (see Table 5).

Example 16a(2) Drugs/Solutions for the Mouse Endotoxemia Study

The drugs/solutions used in the mouse endotoxemia study are thefollowing (listed from (A) to (H)).

(A) Protein formulation buffer (FB)-: This buffer is the negativecontrol within the study and comprises 50 mM Tris-HCl (pH 7.6)+150 mMNaCl (<0.02 EU/ml). This buffer is to be used as a test article anddiluent for positive control and test samples.

(B) Ala-Cpn10: This Cpn10 polypeptide is the positive control within thestudy and has a stock concentration of 5 mg/ml (<0.01 EU/mg). A 1 mg/mlworking solution was made by diluting 400 μl of the protein solutioninto 1.6 ml of formulation buffer.

(C) Ala-Cpn10-Δml: This Cpn10 polypeptide has a stock concentration of3.5 mg/ml (<0.03 EU/mg). A 1 mg/ml working solution was made by diluting571 μl of the protein solution into 1.429 ml of formulation buffer.

(D) Ala-Cpn10-Δroof: This Cpn10 polypeptide has a stock concentration of4.2 mg/ml (<0.1 EU/mg), A 1 mg/ml working solution was made by diluting477 μl of the protein solution into 1.523 ml of formulation buffer.

(F) X-Cpn10: This Cpn10 polypeptide has a stock concentration of 5 mg/ml(<0.04 EU/mg), A 1 mg/ml working solution was made by diluting 400 μl ofthe protein solution into 1.6 ml of formulation buffer.

(G) Endotoxin: Lipopolysaccharide (LPS) was obtained Sigma ChemicalCompany (Cat. No. L6529) Immediately prior to use, vial contents (1 mg)were reconstituted in 1 ml sterile saline. Contents were further diluted(1/10) to 100 μg/ml in sterile saline prior to injection of each group.

(H) Endotoxin Control: Sterile saline for injection was obtained fromPfizer, Australia (Cat. No. DW-SC0010) at a concentration of 900 mg/ml(0.9%) (<0.01 EU/ml).

Example 16b Drug Administration and Blood Collection for the MouseEndotoxemia Study

The protocol for the drug administration in different groups of mice isas shown in Table 5 (see below). All administrations were carried outvia tail vein injections of 100 μl volumes on conscious, restrainedmice. All LPS doses were 10 μg/mouse. All Cpn10 variants were injectedat 100 μg/mouse (100 μL of volume).

The protocol for blood collections is outlined below in Table 5, Bloodsamples were collected via cardiac puncture under halothane anaesthesia(Zeneca Ltd., Macclesfield, UK) (SOP ET-011) or via post-mortemopen-heart bleed. The blood was collected into paediatric serum tubeswith no anticoagulant (clot activator), (Greiner-bio-one, USA,Cat#450401). The samples were left at room temperature for approximately5 min to improve coagulation prior to centrifugation at 12000 rpm for 5min (Biofuge 13, Heraeus Instruments) at room temperature. The serum wastransferred to a fresh tube and placed at −20° C. prior to shipping ondry ice.

All drug administration and blood collection times were recorded on theclinical record sheets (Form IMVS 2061/A). The same record sheets wereuse to monitored general condition throughout the course of theexperiment.

TABLE 5 Protocol for Cpn10 Endotoxemia study. Mice Blood identity Drugadministration times (T) collection time Groups numbers T = 0 min T = 30min T = 2 hr 1 1-7 FB LPS All 2  8-14 FB Saline All 3 15-21 Ala-Cpn10LPS All 4 22-28 Ala-Cpn10 Saline All 5 29-35 Ala-Cpn10-Δml LPS All 636-42 Ala-Cpn10-Δml Saline All 7 43-49 Ala-Cpn10-Δroof LPS All 8 50-56Ala-Cpn10-Δroof Saline All 11 71-77 X-Cpn10 LPS All 12 78-84 X-Cpn10Saline All

Example 16c Cytometric Bead Array (CBA) Analysis

Mouse inflammation CBA analysis (Cat#552364, BD Biosciences) wasperformed on serum samples to assess for changes in the level ofinflammation associated cytokines (i.e. TNFα, IL6, IL-10, MCP-1,IL12p70, IFN-γ Sera from LPS-challenged mice (Table 5, groups 1, 3, 5,7, 9 and 11) or Saline control mice (Table 5, groups 2, 4, 6, 8, 10 and12) were diluted prior to analysis In assay diluent as appropriate (1 in5 for LPS treated groups and 1 in 2 for saline controls). Each samplewas analyzed in duplicate as per manufacturer's instruction using BDFACS-Array instrument with the CBA software.

Example 16d Percent Cytokine Level Reduction Measurement

The following formula was used to determine the effect of Cpn10treatment on LPS challenged mice. The percent reduction of LPS-inducedcytokine in mice pretreated with Cpn10 variants (i.e. Experimental)relative to non-pretreated mice (i.e. Control) was calculated accordingto the following formula: % reduction=100−[(mean cytokine level ofExperimental/mean cytokine of Control)×100]

Example 16e ELISA Analysis

Mouse TNF-α ELISA was carried out using RnD Systems Duoset ELISA kit(Cat#DY410) as per manufacturer's instruction to confirm the CBAanalysis. Dilutions of the samples (1 in 3 dilutions) and standards wereperformed using PBS+10% FCS as diluent. Samples were analyzed induplicate.

Example 16f Clinical Observations

The behavior of the various groups of mice described in Table 5, wasexamined during the 90 min period after LPS or Saline injection andprior to bleeding via cardiac puncture. All observations were recordedand summarized in Table 6. Observations were made immediately afterLPS/saline injection and prior to bleeding of mice by Cardiac Puncture(C.P.). Comparisons of clinical observations were made relative togroups 1, 2or 3. In general, mice treated with LPS alone (Group 1)showed the effects of LPS-induced sepsis within 15 min of injection.LPS-treated mice demonstrated reduced mobility and were less responsiveto stimuli (e.g. noise or touching). Some typical adverse effects due toLPS challenge such as diarrhea or ruffled fur was not observed in any ofthe mice in this study. This may reflect the relative potency of the lotnumber of LPS used on this occasion. Saline treated control mice (Group2) were more responsive to stimulus, exhibiting normal reactions andmobility.

Mice which were pre-treated with Cpn10 and various Cpn10 mutantsdisplayed slightly different behavior in response to LPS challenge,relative to the untreated LPS control group. Mice pre-treated withAla-Cpn10 and Ala-Cpn10-Δroof (Groups 3 and 7) appeared less subdued,slightly more alert and responsive to stimulus but continued to huddletogether for most of the period of observation. Interestingly, micepre-treated with Ala-Cpn10-Δml (Group 5) were slightly more active anddid not huddle as much throughout the observation period. The effect ofLPS challenge in mice pre-treated with Acetyl-Cpn10 (Group 9) alsoappeared different to those observed in other groups. Although thesemice showed similar behavior to Group 3 in the first 30-45 min after LPSinjection, these mice appeared to recover towards the end of theobservation period and displayed increased mobility and alertness. Incontrast, mice pre-treated with X-Cpn10 showed very similar behavior toGroup 1. These mice were very much inactive, less responsive to stimulusand huddled together for most of the observation period. The salinecontrols for each of the Cpn10 mutant groups (i.e. Groups 4, 6, 8, 10and 12) demonstrated an absence of adverse clinical symptoms similar tocontrol animals in the untreated saline group (Group 2).

The bleeding of mice challenged with LPS was typically more problematicas these mice are generally hypotensive (a sepsis-associated symptom).Cardiac puncture bleeds in these mice were slower with reduced recoveryof blood volume relative to mice which did not receive LPS. For micewhich were not able to be bled by direct cardiac puncture, a post-mortemopen heart bleed was performed. Cpn10 treatment did not appear to affector improve blood recovery in LPS treated mice however, in comparison tothe LPS control animals, it was noted that blood from Cpn10-treated micecould generally be recovered by direct cardiac puncture withoutresorting to an open heart bleed. At least 400 μl of blood was recoveredfrom each mouse in this study. All deviations from a normal bleed andrecovery were noted on the relevant clinical record sheet.

TABLE 6 Clinical observation of mice pre-treated with various Cpn10followed with LPS/Saline injection. Group #. Treatment BehaviourClinical signs 1. FB + LPS Not active No ruffled fur No response tostimuli, or diarrhea Huddling Slower/difficult bleeding 2. FB + salineActive and alert Normal bleeding Responsive to stimuli 3. Ala-Cpn10 +LPS Reduced activity Normal bleeding Reduce response to stimuli Huddling4. Ala-Cpn10 + saline Active and alert Normal bleeding Responsive tostimuli 5. Ala-Cpn10-Δml + LPS Reduced activity Normal bleeding Reducedresponse to stimuli Reduced huddling 6. Ala-Cpn10-Δml + saline Activeand alert Normal bleeding Responsive to stimuli 7. Ala-Cpn10-Δroof + LPSLess active Some difficult Less response to stimuli bleeding Huddling 8.Ala-Cpn10-Δroof + Active and alert Normal bleeding saline Responsive tostimuli 11. X-Cpn10 + LPS Not active, Reduced No ruffled fur response tostimuli or diarrhea Huddling Slower/difficult bleeding 12. X-Cpn10 +saline Active and alert Normal bleeding Responsive to stimuli

Example 16g Reduction of Cytokines in Mice

The mean level of TNF-α, IL-6 and IL-10 cytokines in LPS-challenged mice(see FIG. 15) is shown. Cpn10-treated groups demonstrating statisticalsignificance in the reduction of pro-inflammatory cytokines relative toanimals which were not treated with Cpn10 are indicated by asterisk. Thepercentage reduction in the various cytokines analyzed in micepre-treated with various Cpn10s relative to non-pretreated mice (asshown in table 5) are indicated in brackets.

TABLE 7 Mean and percent reduction of LPS-induced pro-inflammatorycytokines in LPS- challenged mice treated with various Cpn10 mutants.Mean and percentage reduction of serum cytokines Pre-treatment TNF-αIL-6 IL-10 None 2033 5667 808 Ala-Cpn10 1337 (34%) 2394* (57%) 365 (54%)Ala-Cpn10-Δml 746* (63%)  4105 (27%) 539 (33%) Ala-Cpn10-Δroof 1010(50%) 3052* (46%) 498 (38%) X-Cpn10 969.3* (52%)   2769* (51%) 469 (42%)CBA Analysis for Serum Cytokine Levels

Serum samples from each mouse were analyzed using the BD Mouseinflammation CBA assay for the detection of circulating pro-inflammatorycytokines. The assay detects TNFα, IL-6, IL-10, IL-12p70, MCP-1 andIFN-γ cytokines in the test sample. The analyses were performed onsamples which were serially diluted (see example 16a) for optimaldetection within the limits of the assay. All samples were analyzed induplicate.

The relative expression of the cytokines TNF-α, IL-6 and IL-10 incontrol versus Cpn10-treated animals as indices of inflammation in thismouse sepsis model, was examined. In the time frame of this endotoxemiastudy (90 min LPS administration), levels of IFNγ, MCP-1 and IL-12p70cytokines are generally outside the detection limits of this assay andtherefore the data is not presented here. As such, these cytokines werenot examined in this study. Prior to analysis of the CBA results, weassessed the robustness of the data based on consistency betweenreplicates and where the data point fell with respect to the linearrange of the standard curve. Extreme outliers were excluded from theanalysis.

The absolute value and mean of circulating TNF-α, IL-6 and IL-10 in thevarious groups of mice in this study are shown in FIG. 15. As expectedthe mean level of TNF-α, IL-6 and IL-10 cytokines were higher in seraderived from mice challenged with LPS relative to their saline controls(FIGS. 15A, C, E vs B, E, F respectively). This indicates that theamount of LPS used in this study induced an inflammatory response andthat the background levels of these cytokines were sufficiently low inthese mice. In some saline control samples (FIG. 15F, ‘X-Cpn10+Saline’)the level of IL-10 detected was as high as to what was detected InLPS-challenged control (i.e. Group 1-‘FB+LPS’). These samples wereanalyzed in a more concentrated form and thus endogenous serum factorsmay have interfered with the assay readout thus contributing toinaccuracies with this data. The data also show that mice challengedwith LPS have reduced serum TNF-α, IL-6 and IL-10 cytokine levels whenpre-treated with any of the Cpn10 proteins compared to non-Cpn10pre-treated mice (FIGS. 15A, C and E, respectively). For each cytokineprofile, a one-way ANOVA analysis (with Tukey's post-hoc test) wasperformed. The analysis found that some of the means of TNF-α and IL-6level but not IL-10 cytokine level in the various groups arestatistically different (FIGS. 15A, C and E). The lack of statisticaldifference in the IL-10 cytokine level despite the apparent reduction ofmean cytokine level shown on the plot is likely due to the largevariation of the IL-10 cytokine in the control group alone (FIG. 15E,‘FB+LPS’). A larger sampling population may improve the statisticalsignificance of these observations for future studies.

Tukey's post-hoc tests showed that the mean TNF-α and IL-6 cytokinelevels in groups pre-treated with either Ala-Cpn10-Δroof or X-Cpn10(i.e. Groups 7 and 11) was considered to be statistically lower relativeto the untreated group (i.e. Group 1) (Table 7). However, in somegroups, a statistically significant reduction in pro-inflammatorycytokines (relative to the untreated group) was only seen in one of thecytokine profiles. For example, Ala-Cpn10-Δml pre-treated group had areduced mean TNF-α but not IL-6 cytokine levels relative to theuntreated group and vice versa for Ala-Cpn10 pre-treated group (Table7). Furthermore, statistical analysis did not find the mean cytokinelevels of the Cpn10 variant pre-treated groups relative to each other tobe different.

Despite statistical significant reduction in only some of the Cpn10variant pre-treated group, the overall trend showed that all Cpn10variants reduced inflammatory associated cytokines in LPS-challengedmice. Overall approximately 30 to 50% reduction of TNF-α, IL-6 and IL-10cytokines level in these mice were observed (Table 7).

Discussion and Conclusions

This study showed that pre-treatment of mice with various Cpn10polypeptides (i.e. Ala-Cpn10, Ala-Cpn10-Δml, Ala-Cpn10-Δroof, andX-Cpn10) appeared to reduce clinical effects of LPS-induced endotoxemia.Mice which received LPS alone displayed typical symptoms of endotoxemia(i.e. reduced activity, responsive and alertness). On the other hand,mice pre-treated with any of the Cpn10 proteins appeared less affectedby LPS challenge (i.e. more responsive and mobile to stimulus) (Table6).

CBA analysis for inflammation associated cytokines showed that the seraof all Cpn10 variant pre-treated mice prior to LPS injection havereduced levels of TNF-α, IL-6 and IL-10 cytokines relative to sera ofmice which received LPS alone. As expected, mice pre-treated withAla-Cpn10 showed reduced levels of TNF-α and IL-6 cytokines, consistentwith our previous results (Johnson et al, 2005). The current resultssuggest that Ala-Cpn10 may reduce IL-10 production in response to allTLR agonists. We next established a similar reduction in the levels ofthese inflammation associated cytokines in mice pre-treated with anumber of Cpn10 variants (Table 7). Although, statistical analysis showthe reduction of TNF-α and IL-6 cytokine levels is only significant withsome of the Cpn10 variant pre-treatment relative to untreated group(FIGS. 15A&C, Table 7), the overall trend shows that all Cpn10 proteinsused in this study appear to down modulate the LPS-inflammatoryresponse.

The in vivo endotoxemia results reveal that all Cpn10 polypeptidesstudied (i.e. Ala-Cpn10, Ala-Cpn10-Δml, Ala-Cpn10-Δroof, and X-Cpn10)have similar activities to Ala-Cpn10. This reconfirms that the mobileloop or the roof loop regions of Cpn10 are not necessary for modulationof the response to TLR4-LPS ligation. The variations observed in the invitro activities of X-Cpn10 could not be assessed in the endotoxaemiastudy as the mean cytokine levels between Cpn10 variant pre-treatedgroups are not statistically different.

Example 17 Purification of Cpn10 from E. coli Batch Fermentation

A bioprocess has been developed for the production of Cpn10polypeptides. As demonstrated below, this process has successfully beenused for the production of ˜20 g of recombinant human Cpn10 from a 100liter E. coli fermentation with >99% purity, ≦0.03 EU mg⁻¹ endotoxin and≦155.3 pg mg⁻¹ DNA. The process is outlined below.

Fermentation

A vial containing the E. coli strain XL1-Blue harboring theAlaCpn10_pPL550 plasmid was retrieved from the master cell bank (see‘General Methods’ above) and ‘pre-cultured’ overnight in Soya Broth withno antibiotic supplementation at 30° C. An inoculum culture wassubsequently prepared for the 100 L bioreactor maintaining the abovemedia and growth temperature parameters. An aliquot of this inoculum wasdispensed into a 100 L bioreactor in a soya-based peptone-enrichedminimal media containing no animal-derived products (BresaGen, SA,Australia) and no antibiotic supplementation. The bioreactor cultivationdid not require batch feeding and the temperature was maintainedthroughout the growth phase at 30±0.1° C. The pH was maintained at7.0±0.2 by the addition of ammonia. Induction of Cpn10 was achieved by atemperature shift to 42° C. at an OD600 of 10 and further incubation at42±0.1° C. for 3 h at which time the fermentation reached an OD600 ofbetween 20-25.

Cell Lysis and Preparation of a Soluble Lysate

Bacterial cells (˜3.5 kg wet weight) were pelleted by centrifugation(5,000×g), resuspended in 25 mM Tris-HCl (pH 8.0) and lysed by 3passages through an APV Gaulin Model 30CD pressure homogenizer at 7000psi (APV, USA) within three hours of the completion of fermentation. Thesoluble Cpn10 contained in the bacterial lysate was harvested afterpelleting cell debris using a flow-through Westfalia MSB-7 centrifuge.Approximately 20 L of clarified lysate was stored overnight at 4° C.

Purification of Cpn10

A three-step downstream process was developed for the purification ofCpn10, incorporating Big Bead Sulfopropyl-Sepharose (BBSP) cationexchange, Q-Sepharose Fast Flow (QFF) anion exchange and HighPerformance Sepharose SP(HPSP) cation exchange chromatography.Chromatography was carried out using a K-prime 40-II Bioprocess Unit(Millipore).

De-pyrogenation of Chromatography Columns, Containers and Buffers

All ion-exchange chromatography columns were depyrogenated by washingwith 1M NaOH and equilibrated with buffer until eluates returned to thepH and conductivity of the specific buffers. All containers used forbuffer storage and receipt of column eluates in the various purificationsteps were pyrogen-free. All buffers used in the purification wereproduced using Water For Injection (WFI).

Big Bead Sulfopropyl-Sepharose Chromatography

The lysate was loaded over 20-40 min onto a BBSP cation exchange column(BPG 300/500 with 8.6 L of BBSP SP-Sepharose resin, GE Biosciences)pre-equilibrated with 25 mM Tris-HCl, pH8.0 (Buffer A), at 75 cm hr⁻¹linear flow-rate and a loading rate of up to 10 g Cpn10 per liter ofresin. After washing with Buffer A, the captured Cpn10 was eluted withBuffer A containing 150 mM NaCl and 1 L fractions were collected.SDS-PAGE analysis showed the BBSP pool to be >95% pure.

Q-Sepharose Fast Flow Chromatography

The BBSP eluate was desalted against two changes of 15 volumes of BufferA at room temperature. The first dialysis step was performed for 2-3 hr,followed by transfer to a fresh Buffer A tank where dialysis continuedovernight. The redistribution of NaCl from the dialysis bags into thetank buffer was monitored by measuring dialysate conductivity (Cyberscan100, Eutech Instruments, Singapore) against calibrated standards. Thedialyzed BBSP pool was loaded onto a BPG 200/500 column packed with 4.7L of QFF anion exchange resin (GE Biosciences) pre-equilibrated inBuffer A at a linear flow-rate of 75 cm hr⁻¹ and a loading rate of up to15 g Cpn10 per liter of resin. The QFF anion exchange flowthroughcontaining the Cpn10 was collected. Under the loading conditions, themajority of the E. coli cell proteins, nucleic acid and endotoxinremained bound to the matrix.

Sulfopropyl-Sepharose High Performance Chromatography

The QFF flowthrough was applied to a BPG 100/500 column packed with 1.67L of SPHP resin (GE Biosciences), pre-equilibrated with 50 mM Tris-HClpH 7.6 (Buffer B), at 15-20 g Cpn10 per liter of resin. The bound Cpn10was eluted with a linear gradient from 0-120 mM NaCl over 15 L and 0.5 Lfractions were collected. The fractions were pooled according to sizeexclusion chromatography (SEC), HPLC and SDS-PAGE analyses. The Cpn10protein and NaCl Ion concentrations were determined by UV Absorbance at280 nm and the Ion Selective Electrode method (IDEXX, Australia),respectively.

Formulation

Based on Na⁺ and Cl⁻ ion measurements combined with conductivitymeasurements of the pooled SPHP fractions, the buffer was adjusted to afinal formulation of 50 mM Tris-HCl pH 7.6 containing 150 mM NaCl. Theformulated Cpn10 was filtered through a 0.2 μm filter under asepticconditions. The filtered solution was dispensed into 500 ml pyrogen-freeplastic bottles, with each bottle receiving 500 ml for a total of 2.5 gCpn10 per bottle.

SDS-PAGE Analysis

SDS PAGE analyses on E. coli cell lysates and chromatography fractionswas performed using NuPAGE 4-12% Bis-Tris gradient gels (Invitrogen,Carlsbad, Calif.) according to the manufacturer's protocol. Gels wereCoomassie Brilliant Blue stained and each gel included Cpn10 protein andmolecular weight standards.

Quantitation of Purified Cpn10 by Spectrophotometry

Purified Cpn10 concentrations were determined by UV absorbance (BioRadSmartSpec-3000 spectrophotometer) at 280 nm using an extinctioncoefficient of 0.353 mg⁻¹ mL⁻¹ cm⁻¹. It should be noted that the BioRadSmartSpec-3000 typically return an A_(280 nm) value of 0.59 for bovineserum albumin (Pierce) while the literature value is 0.67 (Piercetechnical resource No. TR0006.0).

Summary of Cpn10 purity and yields throughout the above describedpurification process are shown in Table 8, in (A) total proteinconcentrations were determined by the BCA protein assay (Sigma) andCpn10 purity was determined by densitometry of cell lysate andCpn10-containing fractions after each purification step. (B) Comparisonof the final purity and yields from three Cpn10 purifications. Eachpurification was prepared from a 100 L E. coli batch fermentation. Allpreparations had a final Cpn10 purity of >99% by Coomassie stainedSDS-PAGE. Endotoxin units (EU) are expressed as EU per mg of Cpn10 whileDNA levels are expressed as pg per mg of Cpn10.

TABLE 8A Total Cpn10 Total Cpn10 Step Protein Volume purity yieldPurification Steps (g) (L) (%) (%) E. coli soluble lysate 95 20.8 — —Big Bead Sulfopropyl-Sepharose 28.8 6.2 >98 30 Q-Sepharose Fast Flow26.1 8.6 >99 91 Sulfopropyl-Sepharose High 21.3 2.1 >99 82 Performance

TABLE 8B Cpn10 Total Cpn10 Endotoxin Host DNA Batch No. (g L⁻¹) (g) (EUmg⁻¹) (pg mg⁻¹) 1 4.84 20.6 0.02 <122.9 2 5.03 16.0 <0.01 <155.3 3 4.9622.9 0.03 <133.3

Example 18 Compositions

Molecules and agents of the present invention, and those identified bymethods of the invention may be used for the treatment or prevention ofvarious disease states and conditions. Such molecules and agents may beadministered alone, although it is more typical that they beadministered as a pharmaceutical composition.

In accordance with the best mode of performing the invention providedherein, specific preferred compositions are outlined below. Thefollowing are to be construed as merely illustrative examples ofcompositions and not as a limitation of the scope of the presentinvention in any way.

Example 18(a) Composition for Parenteral Administration

A composition for intramuscular injection could be prepared to contain 1mL sterile buffered water, and 1 mg of a suitable agent or molecule.

Similarly, a composition for intravenous Infusion may comprise 250 ml ofsterile Ringer's solution, and 5 mg of a suitable agent or molecule.

Example 18(b) Injectable Parenteral Composition

A composition suitable for administration by injection may be preparedby mixing 1% by weight of a suitable agent or molecule in 10% by volumepropylene glycol and water. The solution is sterilized by filtration.

Example 18(c) Capsule Composition

A composition of a suitable agent or molecule in the form of a capsulemay be prepared by filling a standard two-piece hard gelatin capsulewith 50 mg of the agent or molecule, in powdered form, 100 mg oflactose, 35 mg of talc and 10 mg of magnesium stearate.

Example 18(d) Eye Prop Composition

A typical composition for delivery as an eye drop is outlined below:

Suitable agent or compound 0.3 g Methyl Hydroxybenzoate 0.005 g PropylHydroxybenzoate 0.06 g Purified Water about to 100.00 ml.

The methyl and propyl hydroxybenzoates are dissolved in 70 ml purifiedwater at 75° C., and the resulting solution is allowed to cool. Thesuitable agent or molecule is then added, and the solution sterilized byfiltration through a membrane filter (0.22 μm pore size), andaseptically packed into sterile containers.

Example 18(e) Composition for Inhalation Administration

For an aerosol container with a capacity of 20-30 ml: a mixture of 10 mgof a suitable agent or compound with 0.5-0.8% by weight of a lubricatingagent, such as polysorbate 85 or oleic acid, is dispersed in apropellant, such as freon, and put into an appropriate aerosol containerfor either intranasal or oral inhalation administration.

Example 18(f) Ointment Composition

A typical composition for delivery as an ointment includes 1.0 g of asuitable agent or molecule, together with white soft paraffin to 100.0g, dispersed to produce a smooth, homogeneous product.

Example 18(g) Topical Cream Composition

A typical composition for delivery as a topical cream is outlined below:

Suitable agent or molecule 1.0 g Polawax GP 200 25.0 g Lanolin Anhydrous3.0 g White Beeswax 4.5 g Methyl hydroxybenzoate 0.1 g Deionised &sterilised Water to 100.0 g

The polawax, beeswax and lanolin are heated together at 60° C., asolution of methyl hydroxybenzoate is added and homogenization achievedusing high speed stirring. The temperature is then allowed to fall to50° C. The agent or molecule is then added and dispersed throughout, andthe composition is allowed to cool with slow speed stirring.

Example 18(h) Topical Lotion Composition

A typical composition for delivery as a topical lotion is outlinedbelow:

Suitable agent or molecule 1.2 g Sorbitan Monolaurate 0.8 g Polysorbate20 0.7 g Cetostearyl Alcohol 1.5 g Glycerin 7.0 g Methyl Hydroxybenzoate0.4 g Sterilised Water about to 100.00 ml

The methyl hydroxybenzoate and glycerin are dissolved in 70 ml of thewater at 75° C. The sorbitan monolaurate, polysorbate 20 and cetostearylalcohol are melted together at 75° C. and added to the aqueous solution.The resulting emulsion is homogenized, allowed to cool with continuousstirring and the agent or molecule is added as a suspension in theremaining water. The whole suspension is stirred until homogenized.

1. An isolated Cpn10 polypeptide, wherein a glycine residue replaces anextra N terminal alanine residue of said polypeptide as compared to acorresponding wild-type Cpn10 polypeptide, wherein said polypeptidecomprises the amino acid sequence as set forth in SEQ ID NO:30.
 2. Anisolated nucleic acid molecule having a nucleotide sequence encoding aCpn10 polypeptide comprising the amino acid sequence as set forth in SEQID NO: 30, wherein said nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 4, 5, 7, 8, 10, 25, 27, 29, 31 and
 43. 3. Anexpression construct comprising the nucleic acid molecule of claim 2,optionally operably-linked to one or more regulatory sequences.